Efficient delivery of therapeutic molecules in vitro and in vivo

ABSTRACT

Compositions are described for direct protein delivery into multiple cell types in the mammalian inner ear. The compositions are used to deliver protein(s) (such as gene editing factors) editing of genetic mutations associated with deafness or associated disorders thereof. The delivery of genome editing proteins for gene editing and correction of genetic mutations protect or restore hearing from genetic deafness. Methods of treatment include the intracellular delivery of these molecules to a specific therapeutic target.

FIELD OF THE INVENTION

The present invention relates to protein therapeutics including genome-editing. Embodiments are directed to delivery of proteins that are fused to an anionic molecule, antimicrobial or membrane destabilization molecules. These anionic molecules include, an oligonucleotide, a polynucleotide, negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids.

BACKGROUND

Therapeutic proteins including peptide hormones, cytokines, and monoclonal antibodies have achieved widespread success as research tools and are among the fastest growing classes of drugs. Many powerful and potentially therapeutic proteins have been discovered or engineered over the past two decades, including enzymes capable of metabolic complementation (Hartung, S. D. et al. Gene. Mol. Ther. 9, 866-875 (2004)), neutralizing antibodies against intracellular targets (Wang, J. et al. Nat. Biotechnol. 26, 901-908 (2008)), engineered transcription factors (Urnov, F. D., et al. Nat. Rev. Genet. 11, 636-646 (2010)), and programmable genome-editing enzymes (Sander, J. D. & Joung, J. K. Nat. Biotechnol. 32, 347-355 (2014); Gaj, T., et al. Trends Biotechnol. 31, 397-405 (2013)). While protein biologics have proven effective for extracellular targets, their use to address intracellular targets is comparatively undeveloped due to the inability of most proteins to spontaneously enter mammalian cells. Enabling exogenous proteins to access intracellular targets is most commonly achieved by delivery of their encoding DNA sequences through chemical transfection (Midoux, P., et al. Br. J. Pharmacol. 157, 166-178 (2009)), electroporation (Bodles-Brakhop, A. M., et al. Mol. Ther. 17, 585-592 (2009)), or viral delivery (Kay, M. A., et al. Nat. Med. 7, 33-40 (2001)). The introduction of exogenous DNA into cells, however, raises the possibility of permanent recombination into the genome, potential disruption of endogenous genes, and long-term exposure to the encoded agent. For some research or therapeutic applications, including genome editing applications that seek to effect a one-time, permanent modification of genomic DNA, the functional delivery of non-replicable protein agents may offer improved safety or broader applicability.

The recent development of methods to deliver in vitro transcribed mRNAs or mRNA analogs has offered an alternative to DNA delivery without requiring nuclear transport of an encoding gene, and with greatly reduced potential for genomic insertion of the foreign nucleic acid. While promising, mRNA delivery continues to face challenges including immunogenicity and RNA stability. While chemical modifications and the inclusion of base analogs can mitigate some of these issues, the large-scale production of high-quality modified mRNAs remains a challenge (Zangi, L. et al. Nat. Biotechnol. 31, 898-907 (2013)). Moreover, proteins containing important natural or synthetic post-translational modifications may not be amenable to production by endogenous translation machinery. Therefore, while both DNA and mRNA delivery have become powerful research tools with therapeutic implications, the development of effective and general protein delivery methods remains an important challenge for the molecular life sciences.

Current or conventional protein delivery technologies are based on fusion or conjugation to cationic molecules that facilitate endocytosis, such as unstructured peptides (Wadia, J. S., et al. Nat. Med. 10, 310-315 (2004); Daniels, D. S. & Schepartz, A. J. Am. Chem. Soc. 129, 14578-14579 (2007)) or engineered superpositively charged proteins (Cronican, J. J. et al. ACS Chem. Biol. 5, 747-752 (2010); Thompson, D. B., et al. Methods Enzymol. 503, 293-319 (2012); Thompson, D. B., et al. Chem. Biol. 19, 831-843 (2012)). While such delivery can be effective in cell culture, and has even shown some success in vivo, cationic protein-based delivery methods have not seen widespread adoption. Unprotected proteins can be rapidly degraded by extracellular and endosomal proteases (Heitz, F., et al. Br. J. Pharmacol. 157, 195-206 (2009)), or neutralized by binding to serum proteins, blood cells, and the extracellular matrix (Caron, N. J. et al. Mol. Ther. J. Am. Soc. Gene Ther. 3, 310-318 (2001); Chesnoy, S. & Huang, L. Annu. Rev. Biophys. Biomol. Struct. 29, 27-47 (2000)). In addition, the low efficiency of endosomal escape and avoidance of lysosomal degradation are major challenges to all endocytic protein delivery strategies, as evidenced by ongoing interest in endosome altering (Thompson, D. B., et al. Chem. Biol. 19, 831-843 (2012); Al-Taei, S. et al. Bioconjug. Chem. 17, 90-100 (2006)) and destabilizing strategies (Shete, H. K., J. Nanosci. Nanotechnol. 14, 460-174 (2014)). These challenges have proven especially difficult in vivo (Aguilera, T. A., et al. Integr. Biol. Quant. Biosci. Nano Macro 1, 371-381 (2009)).

Nucleic acid delivery has benefited greatly from the development of liposomal reagents over the past two decades. Cationic lipid formulations have enabled DNA and RNA transfection to become a routine technique in basic research and have even been used in clinical trials (Coelho, T. et al. N. Engl. J. Med. 369, 819-829 (2013)). The lipid bilayer of the vehicle protects encapsulated nucleic acids from degradation and can prevent neutralization by antibodies (Judge, A. D., et al. Mol. Ther. J. Am. Soc. Gene Ther. 13, 494-505 (2006)). Importantly, fusion of liposomes with the endosomal membrane during endosome maturation can enable the efficient endosomal escape of cationic lipid-delivered cargo (Basha, G. et al. Mol. Ther. J. Am. Soc. Gene Ther. 19, 2186-2200 (2011)). More advanced reversibly ionizable lipid nanoparticles enable efficient encapsulation and delivery of nucleic acids, while avoiding non-specific electrostatic interactions and sequestration (Semple, S. C. et al. Nat. Biotechnol. 28, 172-176 (2010)).

Because proteins, in contrast to nucleic acids, are chemically diverse with no dominant electrostatic property, no lipid formulation is likely to drive the efficient delivery of all proteins into mammalian cells. While proteins can be encapsulated non-specifically and delivered by rehydrated lipids in vitro (Boeckle, S., et al. J. Control. Release Off. J. Control. Release Soc. 112, 240-248 (2006); Allen, T. M. & Cullis, P. R. Adv. Drug Deliv. Rev. 65, 36-48 (2013)), the efficacy of encapsulation is dependent on protein concentration, is generally inefficient (Zelphati, O. et al. J. Biol. Chem. 276, 35103-35110 (2001)), and has not seen widespread application. Specialty commercial reagents developed specifically for protein delivery (Adrian, J. E. et al. J. Control. Release Off. J. Control. Release Soc. 144, 341-349 (2010); Morris, M. C., et al. Nat. Biotechnol. 19, 1173-1176 (2001)) have also failed to garner popularity perhaps due to their low potency and unreliability with a variety protein cargoes (Colletier, J.-P., et al. BMC Biotechnol. 2, 9 (2002)).

SUMMARY

Embodiments of the invention are directed to compositions comprising therapeutically effective anionically charged molecules and compositions for their efficient and specific delivery in vitro and in vivo. Targeted cells include any cells associated with hearing, including neurons, stem cells, hair cells, supporting cells and the like.

In an embodiment, a method of treating deafness or disorders thereof, associated with a genetic mutation in a patient in need thereof, comprises administering to an inner ear of the patient a therapeutically effective amount of a chimeric molecule comprising at least one protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The chimeric molecule targets one or more genetic loci associated with deafness in a patient and modulates replication, expression, function or activity of the genetic locus in inner ear cells comprising, strial vascularis, neurons, hair cells, stem cells, or supporting cells. The chimeric molecule comprises a supercharged protein or variants thereof, for example, the supercharged protein is green fluorescent protein (s-GFP), or variants thereof. The gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In an embodiment, the supercharged protein further comprises a membrane destabilization protein, such, as for example, aurein, which is linked to the s-GFP or variants thereof.

In some embodiments the chimeric molecule is encapsulated in a cationic lipid formulation.

In embodiments, the chimeric molecules target different chromosomal abnormalities, such as, for example, deletion, insertion, duplication, inversion and the like. The chimeric molecules are suitable therapeutics where monogenic, bigenic or multigenic mutations contribute to hearing loss. Accordingly, in other embodiments, the chimeric molecules target multiple genes.

In another embodiment, a method of correcting, silencing or modifying a genetic mutation associated with deafness or disorders thereof, in a patient, comprises administering to a patient's inner ear, a cationic liposome encapsulating a therapeutically effective amount of a molecule comprising a supercharged protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate gene expression, protein expression, function, activity or combinations thereof. The molecule targets one or more genetic loci having a mutation associated with deafness or disorders thereof, in a patient, wherein the molecule corrects, silences or modifies a genetic mutation inner ear cells comprising, strial vascularis, hair cells, stem cells, or supporting cells. For gene editing such as CRISPR, Cas9 (protein) is complexed with gRNA (guide RNA, nucleic acid) with/without a template by liposome. Such complex can then be delivered to inner ear for gene editing. For the disruption of mutation, only gRNA without template is needed; whereas for mutation repair, both a gRNA and a template are needed; in addition to Cas9.

In another embodiment, a method of correcting, silencing or modifying a genetic mutation in hair cells, stem cells, and/or supporting cells in vitro or in vivo, comprises contacting a hair cell or supporting cell or administering to a patient's inner ear, a therapeutically effective amount of a molecule comprising a supercharged protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The molecule targets one or more genetic loci having a mutation associated with deafness or disorders thereof, in a patient, wherein the molecule corrects, silences or modifies a genetic mutation in hair cells and supporting cells of the inner ear.

In another embodiment, a method of delivering a therapeutic molecule to cells of an inner ear of a patient, comprises administering to the inner ear of a patient a chimeric molecule comprising at least one protein or peptide fused, complexed or linked to one or more anionic molecules. In an embodiment, the cells of the inner ear comprise hair cells, supporting cells, or combinations thereof. In an embodiment, the chimeric molecule comprises one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate gene expression, protein expression, function, activity or combinations thereof. The gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In an embodiment, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. The oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof. In other embodiments, the chimeric molecule comprises a supercharged protein or variants thereof. An example of a supercharged protein is a fluorescent protein, or variants thereof. The chimeric molecule further comprises a membrane destabilizing protein, for example, aurein.

In some embodiments, the chimeric molecule is encapsulated in a cationic lipid formulation.

In some embodiments, a composition comprises a cationic lipid encapsulating one or more chimeric molecules comprising one or more proteins or peptides fused, complexed or linked to one or more anionic molecules. These one or more anionic molecules confer an overall net negative charge to the chimeric molecule and comprise one or more anionic domains or bind to an anionic nucleic acid domain. In some embodiments, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. In some embodiments, the oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof.

In embodiments, the one or more proteins or peptides are cationic, anionic or are neutrally charged. In some embodiments, the proteins or peptides comprise: enzymes, hormones, chemotherapeutic agents, immunotherapeutic agents, gene editing agents, synthetic molecules or combinations thereof. In some embodiments, the gene editing agents comprise: transcriptional activators, transcriptional repressors, transcription factors, enhancer modulating molecules, recombinases, nucleases, nucleic acid binding-proteins, nucleic acid binding-polynucleotides or oligonucleotides, DNA-binding proteins or DNA-binding nucleic acids, or combinations thereof.

In other embodiments, methods of treatment comprises administering a therapeutically effective amount of a cationic lipid encapsulating one or more chimeric molecules comprising one or more proteins or peptides fused, complexed or linked to one or more anionic molecules.

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B show that after microinjection with Cas9 protein and gRNA complexed with lipofectamine2000, OHCs labeled with Myo7a/DAPI without GFP were seen in the adult Atoh1-GFP transgenic cochlea, a demonstration of genome editing. The efficiency is ˜15%. In control cochlea injected with saline, all cochlear OHCs were positive for GFP, a demonstration of specificity of Cas9:gRNA in genome editing in adult cochlea in vivo.

FIG. 2 is a schematic representation showing a design of gRNA for Cas9:gRNA (SEQ ID NOS: 21-89) injection. Mutation 2765C→T was shown. Multiple gRNAs, some target the mutation and some target wildtype Pmca2 gene were designed for injection with Cas9.

FIG. 3 is a series of graphs showing that Cas9:gRNA injection restored hearing in the Obl heterozygous mice. In the uninjected inner ears, profound hearing loss was shown by the complete lack of ABR and DPOAE across all frequencies at the highest sound pressure level (SPL) from 4 weeks to three months. In the injected inner ear, significant hearing recovery was achieved across most frequencies (except for 45.25 kHz) by ABR as well as by DPOAE. In the middle frequencies 11.32 to 22.64 kHz, the recovery was over 50 dB, a demonstration of restoration of hearing to near normal levels. At 8 weeks and three months of age, hearing recovery was largely maintained shown by ABR and DPOAE. Deterioration in high frequency was seen at later stages.

FIG. 4 are graphs showing that hearing recovery is gRNA specific. An example of a gRNA-2.5-mut with different sequence did not result in hearing improvement after injection with Cas9. gRNAs against wildtype Pmca2 did not improve hearing either.

FIG. 5 shows the high-efficiency cleavage by specific gRNA-2.4-mut. Different concentrations of Cas9 protein and gRNAs were tested for DNA cleavage in vitro. Only gRNA-2.4-mut showed cleavage in mutant but not in wildtype Pmca2 genomic region at 125 nM. Whereas other gRNAs showed cleavages in both mutant and wildtype genomic DNA.

FIG. 6A, 6B show that Cas9:gRNA injection promotes outer hair cell survival. FIG. 6A: Two months after injection, a majority of outer hair cells are seen in the base compared to a complete outer hair cell loss in the uninjected control ear. FIG. 6B: Cell count showed general preservation of outer hair cells across all frequencies after Cas9:gRNA injection two months later. In contrast to almost complete outer hair cell loss at high-frequencies 32 and 45.25 kHz, outer hair cell number in the injected inner ears was virtually intact.

FIGS. 7A, 7B is a color stain showing the preservation of ganglion neurites in the Cas9:gRNA injected cochlea. FIG. 7A: In the control uninjected inner ear of Pmca2^(Obl/+) mice, TuJ labeling of neurites is largely absent in the region between outer hair cells (OHCs) and inner hair cells (IHCs) (Bracket demarcates the region). FIG. 7B: In the injected inner ear in contrast, preservation of neurites is seen by a large number of TuJ⁺ fibers. Parv: parvalbumin that labels hair cells.

FIG. 8 is a series of graphs showing the hearing rescue by Cas9:gRNA in the Bth heterozygous mice. 4 weeks after injection, significant hearing improvement is observed in the middle frequencies of 11.32 to 32.64 kHz in the injected ears comparing to control uninjected inner ears. The DPOAE is similar with the exception at 11.32 kHz whereas injected ears showed elevated DPOAE threshold shift.

FIG. 9 is a graph showing that in four weeks after multiple injections, ABR in the Cas9/gRNA-Pmca2-2.4 injected inner ears show dramatic hearing restoration at 16, 22.64, 32 and 45.24 kHz comparing to the uninjected control ears. Hearing restoration at 16, 22.64 and 32 kHz are improved by 40 dB.

FIGS. 10A and 10B show the in vivo delivery of Cre recombinase to hair cells in the mouse inner ear. FIG. 10A: The scala media (cochlear duct) of P0 floxP-tdTomato mice (n=4) were injected with 0.3 μL of 23 μM (−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5 days, tdTomato expression indicative of Cre-mediated recombination was visualized using immunohistology. Red=tdTomato; green=Myo7a; white=Sox2; blue=DAPI. Yellow brackets indicate the outer hair cell (OHC) region. FIG. 10B: Ten days after (−30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrow), similar to stereocilia in control cochlea. Red=tdTomato; green=Esp, white=Sox2; blue=DAPI, Scale bars: 10 μm.

FIGS. 11A-11E show the high efficiency of protein delivery mediated by aurein in the inner ear in vivo. Injection of (+36)GFP-aurein-Cre with different concentrations to Rosa-tdTf^(/f) mouse cochlea resulted in tdT labeling in cochlear cell types with different efficiencies. FIG. 11A: At 5 μM, tdT labeling was mainly in some IHC and SC. FIG. 11B: At 22.5 μM, tdT labeling was in most IHC and OHC, and in some SC and spiral ganglion neurons (SPN). FIG. 11C: At 50 μM, tdT was seen in most IHC and OHC, as well as a large number of SC. IHC loss was seen under the condition. FIG. 11D: In mice injected with 50 μM (+36)GFP-Cre, very few IHC and HC were labeled with tdT. FIG. 11E: In Opti-MEM injected control mice no tdT labeling was detected in any inner ear cells. Scale bars: 10 μm.

FIG. 12 shows the high efficiency of aurein mediated protein delivery in the mammalian inner ear. Comparison between different concentrations of (+36)GFP-aurein-Cre and control reveled that extremely high-efficient delivery of functional protein (Cre) with the aurein at 22.5 and 50 μM, respectively.

FIG. 13A is a schematic representation showing an overview of protein delivery in mammalian cells. Cationic macromolecules such as +36 GFP interact with anionic sulfated proteoglycans on the cell surface and are endocytosed and sequestered in early endosomes. The early endosomes can acidify into late endosomes or lysosomes. Alternatively, early endosomes may be trafficked back to the cell surface as part of the membrane-recycling pathway. To access the cytoplasm, an exogenous cationic protein must escape endosomes before it is degraded or exported. FIG. 13B is a schematic representation showing the sortase-mediated conjugation of peptides with +36 GFP-Cre recombinase prior to screening. Sortase was used to conjugate synthetic peptides containing a C-terminal LPETGG with expressed +36 GFP-Cre containing an N-terminal GGG. The resulting peptide-LPETGGG-+36 GFP-Cre fusion proteins have the same chemical composition as expressed recombinant proteins, but are more easily assembled.

FIG. 14 is a graph showing a primary screen for cytosolic delivery of Cre recombinase in BSR.LNL.tdTomato cells. Initial screen of 20 peptide-(+36 GFP)-Cre conjugated proteins. Cytosolic Cre delivery results in recombination and tdTomato expression. The percentage of tdTomato positive cells was determined by fluorescence image analysis. 250 nM+36 GFP-Cre was used as the no-peptide control (NP), and addition of 100 μM chloroquine was used as the positive control (+). Cells were treated with 250 nM protein for 4 h in serum-free DMEM. Cells were washed and supplanted with full DMEM and incubated for 48 h. Error bars represent the standard deviation of three independent biological replicates.

FIGS. 15A, 15B are graphs showing the efficacy and toxicity of recombinant expression fusions of aurein 1.2 (“E”) and citropin 1.3 (“U”). FIG. 15A shows that the cytosolic Cre delivery results in recombination and tdTomato expression. The percentage of tdTomato positive cells was determined by flow cytometry. Protein fusions were delivered at 125 nM, 250 nM, 500 nM, and 1 μM. FIG. 15B shows the toxicity of aurein 1.2 and citropin 1.3 as determined by CELLTITERGLO (Promega) assay. Protein fusions were delivered at 125 nM, 250 nM, 500 nM, and 1 μM. The labeled concentration of +36 GFP-Cre was used as the no peptide control (NP), and addition of 100 μM chloroquine was used as the positive control (+). Cells were treated with 250 nM protein for 4 h in serum-free media. Cells were washed and supplanted with full DMEM and incubated for 48 h. Error bars represent the standard deviation of three independent biological replicates.

FIGS. 16A, 16B are graphs showing the activity and cytotoxicity of aurein 1.2 variants fused to +36 GFP-Cre (FIG. 16A). The percentage of tdTomato positive cells was determined by flow cytometry. (FIG. 16B) Toxicity as determined by CELLTITERGLO (Promega) assay. 250 nM+36 GFP-Cre was used as the no peptide control (NP), and addition of 100 μM chloroquine was used as the positive control (+). Cells were treated with 250 nM protein for 4 h in serum-free DMEM. Cells were washed and supplanted with full DMEM and incubated for 48 h.

FIGS. 17A-17D show the ability of +36 GFP and aurein 1.2-+36 GFP dexamethasone-conjugates to reach the cytosol and activate GR translocation. FIG. 17A shows images of HeLa cells expressing GR-mCherry treated in the presence and absence of 1 μM dexamethasone (Dex)-protein conjugates for 30 min at 37° C. FIG. 17B shows that nuclear-to-cytosol GR-mCherry fluorescence ratios (translocation ratios) of respective Dex-protein conjugates determined using CELLPROFILER®. FIG. 17C shows the GR-mCherry translocation ratios resulting from cells treated in the presence and absence of +36 GFP^(Dex) and endocytic inhibitors. FIG. 17D shows the GR-mCherry translocation ratios resulting from cells treated in the presence and absence of aurein 1.2-+36 GFP^(Dex) and endocytic inhibitors. Statistical significance is measured by P-value. ns=P>0.05, *=P≦0.05, **=P≦0.01, ***=P≦0.001.

FIGS. 18A-18C show the results from the in vivo protein delivery of Cre recombinase into mouse neonatal cochleas. 0.4 μL of 50 μM+36 GFP-Cre or aurein 1.2-+36 GFP-Cre were injected into the scala media. FIG. 18A: Five days after injection, cochlea were harvested. Inner hair cells (IHC), outer hair cells (OHC) and supporting cells in the sensory epithelium (SE) were imaged for the presence of tdTomato, which is only expressed following Cre-mediated recombination. Hair cells were labeled with antibodies against the hair-cell marker Myo7a. Gray/white=Myo7a, Red=tdTomato, Blue=DAPI. FIG. 18B: To evaluate cytotoxicity, the number of outer hair cells and inner hair cells were measured by counting DAPI-stained cells. FIG. 18C: The percentage of tdTomato positive cells, reflecting successful delivery of functional Cre recombinase, was determined by fluorescence imaging.

DETAILED DESCRIPTION

Embodiments of the invention are directed to compositions for the efficient intracellular delivery of proteins to the nucleus or cytoplasm. Conventional methods of protein delivery typically rely on cationic peptides or proteins to facilitate endocytosis, but suffer from low tolerance for serum proteins, poor endosomal escape, and limited in vivo efficacy. Herein, it is reported that cationic lipid reagents can potently deliver to hair cells and supporting cells, proteins that are fused to polynucleotides, oligonucleotides, negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids.

All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human.

The following description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application or uses. Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented.

It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements—or, as appropriate, equivalents thereof—and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

As used herein, a “chimeric” molecule is one which comprises one or more unrelated types of components or contain two or more chemically distinct regions or domains which can be conjugated to each other, fused, linked, translated, attached via a linker, chemically synthesized, expressed from a nucleic acid sequence, etc. For example, an antimicrobial or membrane destabilizing peptide and an unrelated peptide, a peptide and a nucleic acid sequence, a peptide and a detectable label, unrelated peptide sequences, unrelated nucleic acid sequence and the like.

The term “anionic” molecule is one which comprises one or more “anionic” domains which confer an overall net anionic charge to the molecule. Accordingly, the chimeric molecule can be an anionic molecule.

A “supercharged” molecule is a molecule e.g. peptide, having a positive or negative charge and when it is connected to another molecule confers an overall positive or negative charge to the entire molecule.

As used herein, a “membrane destabilizing domain” is one which disrupts a cellular membrane in vitro or in vivo, for example, aurein. The term encompasses proteins, peptides, polynucleotides, oligonucleotides, bacterial or viral molecules, antimicrobial peptides (AMP), antibacterial molecules, microtubules, synthetic or natural molecules. A chimeric molecule embodied herein, further comprises one or more membrane destabilizing domains.

As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties or domains are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. The term “connected” will be used for the sake of brevity and is meant to include all possible methods of physically associating each domain of the chimeric molecule to each other. For example, a supercharged protein is typically associated with or connected to a nucleic acid by a mechanism that involves non-covalent binding (e.g., electrostatic interactions). In certain embodiments, a positively charged, supercharged protein is associated with a nucleic acid through electrostatic interactions to form a complex. In some embodiments, a sufficient number of weaker interactions can provide sufficient stability for moieties to remain physically associated under a variety of different conditions. In certain embodiments, the agent to be delivered is covalently bound to the supercharged protein.

As used herein, the term “green fluorescent protein” (GFP) refers to a protein originally isolated from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light or a derivative of such a protein (e.g., a supercharged version of the protein). The amino acid sequence of wild type GFP is as follows: (SEQ ID NO: 20) MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTFSYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK.

Proteins that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homologous are also considered to be green fluorescent proteins. In certain embodiments, the green fluorescent protein is supercharged. In certain embodiments, the green fluorescent protein is superpositively charged (e.g., +15 GFP, +25 GFP, and +36 GFP). In certain embodiments, the GFP may be modified to include a polyhistidine tag for ease in purification of the protein. In certain embodiments, the GFP may be fused with another protein or peptide (e.g., hemagglutinin 2 (HA2) peptide). In certain embodiments, the GFP may be further modified biologically or chemically (e.g., post-translational modifications, proteolysis, etc.).

As used herein, unless otherwise indicated, the terms “peptide”, “polypeptide” or “protein” are used interchangeably herein, and refer to a polymer of amino acids of varying sizes. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or be naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like.

As used herein, a “nucleic acid” or “nucleic acid sequence” or “cDNA” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs, and refers to nucleic acid sequences in which one or more introns have been removed. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, for instance, DNA which is part of a hybrid gene encoding additional polypeptide sequences.

A “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

As used herein, the terms “nucleic acid sequence”, “polynucleotide,” and “gene” are used interchangeably throughout the specification and include complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.

The nucleic acid sequences may be “chimeric,” that is, composed of different regions. In the context of this invention “chimeric” compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide. These sequences typically comprise at least one region wherein the sequence is modified in order to exhibit one or more desired properties.

The term “target nucleic acid” refers to a nucleic acid (often derived from a biological sample), to which the oligonucleotide is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding oligonucleotide directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the oligonucleotide is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context.

In the present context, the terms “nucleobase” covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non-naturally occurring” have subsequently been found in nature. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C₃-C₆)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the “non-naturally occurring” nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. The term “nucleobase” is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.

As used herein, “nucleoside” includes the natural nucleosides, including 2′-deoxy and 2′-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).

“Analogs” in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443, Toulmé, J. J., Nature Biotechnology 19:17-18 (2001); Manoharan M., Biochemica et Biophysica Acta 1489:117-139(1999); Freier S. M., Nucleic Acid Research, 25:4429-4443 (1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic Acid Drug Dev., 10:297-310 (2000)); 2′-O, 3′-C-linked [3.2.0] bicycloarabinonucleosides (see e.g. N. K Christiensen., et al., J. Am. Chem. Soc., 120: 5458-5463 (1998). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

As used herein, “variant” of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

“Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Accordingly, “treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.

The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

As defined herein, a “therapeutically effective” amount of a compound or agent (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or a series of treatments.

As defined herein, an “effective” amount of a compound or agent (i.e., an effective dosage) means an amount sufficient to produce a (e.g., clinically) desirable result.

As used herein, a “pharmaceutically acceptable” component/carrier etc. is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

The terms “determining”, “measuring”, “evaluating”, “detecting”, “assessing” and “assaying” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent.

As used herein, the term “agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the invention at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

By the term “modulate,” it is meant that any of the mentioned activities, are, e.g., increased, enhanced, increased, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values. Modulation can also normalize an activity to a baseline value.

As used herein, the term “kit” refers to any delivery system for delivering materials. Inclusive of the term “kits” are kits for both research and clinical applications. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides or liposomes. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

General Techniques

For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, embryology, and physiology.

With respect to tissue culture and embryonic stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

Supercharged Proteins

Supercharged proteins can be produced by changing non-conserved amino acids on the surface of a protein to more polar or charged amino acid residues. The amino acid residues to be modified may be hydrophobic, hydrophilic, charged, or a combination thereof. Supercharged proteins can also be produced by the attachment of charged moieties to the protein in order to supercharge the protein. Supercharged proteins frequently are resistant to aggregation, have an increased ability to refold, resist improper folding, have improved solubility, and are generally more stable under a wide range of conditions, including denaturing conditions such as heat or the presence of a detergent.

Any protein may be modified using the inventive system to produce a supercharged protein. Natural as well as unnatural proteins (e.g., engineered proteins) may be modified. Example of proteins that may be modified include receptors, membrane bound proteins, transmembrane proteins, enzymes, transcription factors, extracellular proteins, therapeutic proteins, cytokines, messenger proteins, DNA-binding proteins, RNA-binding proteins, proteins involved in signal transduction, structural proteins, cytoplasmic proteins, nuclear proteins, hydrophobic proteins, hydrophilic proteins, etc. A protein to be modified may be derived from any species of plant, animal, and/or microorganism. In certain embodiments, the protein is a mammalian protein. In certain embodiments, the protein is a human protein. In certain embodiments, the protein is derived from an organism typically used in research. For example, the protein to be modified may be from a primate (e.g., ape, monkey), rodent (e.g., rabbit, hamster, gerbil), pig, dog, cat, fish (e.g., Danio rerio), nematode (e.g., C. elegans), yeast (e.g., Saccharomyces cerevisiae), or bacteria (e.g., E. coli). In certain embodiments, the protein is non-immunogenic. In certain embodiments, the protein is non-antigenic. In certain embodiments, the protein does not have inherent biological activity or has been modified to have no biological activity. In certain embodiments, the protein is chosen based on its targeting ability. In certain embodiments, the protein is green fluorescent protein.

Compositions and Treatments

Disclosed herein are compositions for gene editing of cells of the inner ear, and any cell, damaged or otherwise, associated with hearing and hearing loss, such as, for example, stem cells, hair cells, supporting cells, neurites, strial vascularis and the like. In particular, the targeted cells are the hair cells and supporting cells. The compositions, embodied herein, edit mutations associated with deafness or disorders thereof, in hair cells and supporting cells. Hair cells are inner ear sensory cells, including inner hair cells, outer hair cells, and vestibular hair cells. Supporting cells include Deiters cells, Hensen cells, Pillar cells, inner phalangeal cells, inner border cells, Claudius cells, border cells, basal cells, interdental cells, inner sulcus, spiral limbus.

Hearing loss affects a large portion of population yet no treatment is available beyond hearing aids and cochlear implant, both of which provide limited benefits. In the US alone, over 30 millions of people suffer from hearing loss. The major cause of hearing loss in human is due to irreversible loss of the inner ear sensory cells, hair cells, which are responsible for detecting sounds and sensing balance. Further hearing loss can be caused by defective cell types in the inner ear such as strial vascularis and supporting cells, whose regeneration could lead to restoration of hearing.

Accordingly, embodiments are directed to protein-mediated delivery systems to deliver the biological proteins directly to inner ear cell types with functional consequences. With these methods, the proteins delivered have specific functions, and the effect is transient. Further delivery of native protein lessens any potential immune response. Such an approach would not only be valuable for inner ear delivery, but has far reaching effects on the delivery of proteins targeting different diseases, achieving specific effects in multiple organs and cell types.

For efficient protein delivery, proteins need to enter cells with sufficient amount, and are released from endosomes within cells and reach the targets. Improvement in the endosome release could have drastic effects on the amount of protein to reach the target with enhanced biological effect. Antimicrobial or membrane destabilizing peptides, for example, Aurein, in combination with s-GFP, can serve as an effective carrier to deliver functional proteins into mouse inner ear cell types with high efficiency.

High-efficiency delivery of functional proteins in the inner ear has tremendous advantage over conventional systems, including (1). It allows the delivery of unlimited combinations of proteins to mammalian inner ear without limitations on the size or localization of proteins delivered; (2). The effect will be specific; (3). The effect will be transient as delivered proteins will be degraded over time, which allows the modification of proteins (e.g., to reduce the response to degradation pathway) and controls the time length of function. The delivery system can be used to study protein functions, therapeutics for hearing recovery, hearing protection, and gene editing to correct genetic deafness by genome-editing proteins.

In an embodiment, a method of treating deafness or disorders thereof, associated with a genetic mutation in a patient in need thereof, comprises administering to an inner ear of the patient a therapeutically effective amount of a chimeric molecule comprising at least one membrane destabilizing domain and a protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The chimeric molecule targets one or more genetic loci associated with deafness in a patient and modulates replication, expression, function or activity of the genetic locus in hair cells and supporting cells of the inner ear. The chimeric molecule comprises a supercharged protein or variants thereof, for example, the supercharged protein is green fluorescent protein (s-GFP), or variants thereof. The gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In embodiments, the chimeric molecules target different chromosomal abnormalities, such as, for example, deletion, insertion, duplication, inversion and the like. The chimeric molecules are suitable therapeutics where bi-genic or multi-genic mutations contribute to hearing loss. CRISPR technology is well suited to target multiple genes.

In an embodiment, a membrane destabilizing domain comprises one or more of: antimicrobial or membrane destabilizing proteins or peptides, polynucleotides, oligonucleotides, bacterial or viral (e.g. reovirus outer capsid protein or peptide, μ1; papilloma virus capsid protein or peptide L2; etc.), antibacterial molecules, microtubules, lipids, synthetic or natural molecules. Membrane destabilization peptides are known in the art. See, for example, Fernandez, D. I. et al., Biochim. Biophys. Acta, 2009 August; 1788(8):1630-8. Antimicrobial peptides (AMPs) are a class of membrane-active peptides that penetrate microbial membranes to provide defense against bacteria, fungi, and viruses, often with high selectivity (Zasloff, M. Nature 2002, 415, 389).

In an embodiment, the supercharged protein further comprises a membrane destabilization protein, such, as for example, aurein, which is linked to the s-GFP or variants thereof. Accordingly, in one embodiment the chimeric molecule comprises a supercharged molecule connected to a membrane destabilization domain which in turn is connected to one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In another embodiment the chimeric molecule comprises a supercharged molecule connected to a membrane destabilization domain which in turn is connected to a gene editing agent, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In another embodiment the chimeric molecule comprises one or more membrane destabilization domains connected to one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In another embodiment, the chimeric molecule comprises a membrane destabilization domain connected to a supercharged molecule which in turn is connected to one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. One of ordinary skill in the art would understand that there are a multitude of configurations which can be obtained. Accordingly if the letter “S” represents the supercharged domain, the letter “M” represents the antimicrobial or membrane destabilizing domain, and the letter “X” represents the therapeutic molecules responsible for correcting, silencing or modifying a genetic mutation, then the molecular structure of the molecule can be S-M-X, or M-X-X, or S-M-M-X, or S-S-M-M-X, etc. In those cases where there are two or more molecules that comprise a domain, these domains can comprise different molecules or they can be the same molecule. One of ordinary skill in the art would understand the possible configurations that can be possible.

In another embodiment, a chimeric molecule comprises any one or more sequences SEQ ID NOS: 1 to 95 linked to an antimicrobial or membrane destabilization domain. The antimicrobial or membrane destabilization domain can be connected to the supercharged domain on one end and to therapeutic molecules on the other end (e.g. upstream or downstream).

In some embodiments the chimeric molecule is encapsulated in a cationic lipid formulation.

In another embodiment, a chimeric molecule comprises any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide, antimicrobial or membrane destabilizing peptide, gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.

In another embodiment, a chimeric molecule comprises any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide and an antimicrobial or membrane destabilizing peptide and one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.

In another embodiment, a chimeric molecule comprises any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: an antimicrobial or membrane destabilizing domain and one or more supercharged proteins or peptides, gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.

In another embodiment, a chimeric nucleic acid molecule encoding any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide and an antimicrobial or membrane destabilizing peptide, and one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.

In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a chimeric molecule comprises a supercharged protein or peptide and an antimicrobial or membrane destabilizing peptide and one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a chimeric nucleic acid molecule encoding any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprises a supercharged protein or peptide, antimicrobial or membrane destabilizing domain, gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a chimeric molecule comprises a nucleic acid molecule encoding a supercharged protein or peptide and an antimicrobial or membrane destabilizing domain and one or more gene editing agents, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In one embodiment, the nucleic acids are gRNA's. See, for example, FIG. 2, SEQ ID NOS: 21-33 and SEQ ID NOS: 90-95. In one embodiment, the gRNA is SEQ ID NO: 26 (FIG. 2; PMCA2-4mut). In another embodiment, the gRNA comprises SEQ ID NOS: 90-95.

Other examples of chimeric molecules comprising gRNA are

TMC1-1 WT (SEQ ID NO: 90): GGGACAGAACATCCCCAGGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT TMC1-2 WT (SEQ ID NO: 91): GGTGGGACAGAACATCCCCAGGGTTTTAGAGCTAGAAATAGCAAGTTAAA ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT TMC1-3 WT (SEQ ID NO: 92): GGGTGGGACAGAACATCCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT TMC1-1 MUT (SEQ ID NO: 93): GGGACAGAACTTCCCCAGGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT TMC1-2 MUT (SEQ ID NO: 94): GGTGGGACAGAACTTCCCCAGGGTTTTAGAGCTAGAAATAGCAAGTTAAA ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT TMC1-3 MUT (SEQ ID NO: 95): GGGTGGGACAGAACTTCCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT

In another embodiment, the chimeric molecule comprises nucleic acid sequences comprising SEQ ID NOS: 21-33, 90-95 or combinations thereof. In another embodiment, the chimeric molecule comprises SEQ ID NOS: 26, 94, 95 or combinations thereof.

In another embodiment, a method of correcting, silencing or modifying a genetic mutation associated with deafness or disorders thereof, in a patient, comprises administering to a patient's inner ear, a cationic liposome encapsulating a therapeutically effective amount of a molecule comprising a supercharged protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The molecule targets one or more genetic loci having a mutation associated with deafness or disorders thereof, in a patient, wherein the molecule corrects, silences or modifies a genetic mutation in hair cells and supporting cells of the inner ear.

In another embodiment, a method of correcting, silencing or modifying a genetic mutation associated with deafness or disorders thereof, in a patient, comprises administering to a patient's inner ear, a cationic liposome encapsulating a therapeutically effective amount of a molecule comprising a supercharged protein or peptide and an antimicrobial or membrane destabilizing domain fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The molecule targets one or more genetic loci having a mutation associated with deafness or disorders thereof, in a patient, wherein the molecule corrects, silences or modifies a genetic mutation in hair cells and supporting cells of the inner ear.

In another embodiment, a method of correcting, silencing or modifying a genetic mutation in hair cells and/or supporting cells in vitro or in vivo, comprising: contacting a hair cell or supporting cell or administering to a patient's inner ear, a therapeutically effective amount of a molecule comprising an antimicrobial or membrane destabilizing domain, a supercharged protein or peptide fused, complexed or linked to one or more one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The molecule targets one or more genetic loci having a mutation associated with deafness or disorders thereof, in a patient, wherein the molecule corrects, silences or modifies a genetic mutation in hair cells and supporting cells of the inner ear. For gene editing such as CRISPR, Cas9 (protein) is complexed with gRNA (guide RNA, nucleic acid) with/without a template by liposome. Such complex can then be delivered to inner ear for gene editing. For the disruption of mutation, only gRNA without template is needed; whereas for mutation repair, both a gRNA and a template are needed; in addition to Cas9.

In another embodiment, a method of delivering a therapeutic molecule to cells of an inner ear of a patient, comprises administering to the inner ear of a patient a chimeric molecule comprising at least one protein or peptide fused, complexed or linked to one or more anionic molecules. In an embodiment, the cells of the inner ear comprise hair cells, supporting cells, or combinations thereof. In an embodiment, the chimeric molecule comprises one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof. The gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In an embodiment, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. The oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof. In other embodiments, the chimeric molecule comprises a supercharged protein or variants thereof. An example of a supercharged protein is a fluorescent protein, or variants thereof. The chimeric molecule further comprises a membrane destabilizing protein, for example, aurein.

In some embodiments, a composition comprises a cationic lipid encapsulating one or more chimeric molecules. These chimeric molecules comprise one or more proteins or peptides fused, complexed or linked to one or more anionic molecules. The anionic molecules can vary as long as they comprise one or more anionic domains or bind to an anionic nucleic acid domain. It is preferred that the anionic molecules confer an overall net negative charge to the chimeric molecule. Without wishing to be bound by theory, it was hypothesized that proteins that are engineered to be highly negatively charged or that are naturally highly anionic may be able to take advantage of the same electrostatics-driven complexation and encapsulation used by cationic liposomal reagents for nucleic acid delivery. While few proteins natively possess the density of negative charges found in the phosphate backbone of nucleic acids, it was speculated that translational fusion to, or non-covalent complexation with, a polyanionic molecule may render the resulting protein or protein complex sufficiently anionic to be efficiently complexed by common cationic lipid reagents. The results for the work, described in the Examples section which follows, showed that delivery efficiency depends on the net charge of the fusion protein, and natively anionic peptide tags such as 3×FLAG and VP64 can also enable lipid-mediated protein delivery.

Accordingly, in some embodiments, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. In some embodiments, the oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), a short interfering RNA (siRNA), a micro, interfering RNA (miRNA), a small, temporal RNA (stRNA), a short, hairpin RNA (shRNA), mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof.

In embodiments, the one or more proteins or peptides of the chimeric or anionic molecule can possess any charge as long as the overall net charge of the chimeric molecule is anionic. Accordingly, in embodiments, the proteins or peptides are cationic, anionic or are neutrally charged. Examples of proteins or peptides of the chimeric molecule which can be complexed or linked to the polyanionic molecule or domain comprise: enzymes, hormones, chemotherapeutic agents, immunotherapeutic agents, gene editing agents, synthetic molecules or combinations thereof.

In some embodiments, the protein or peptide is a therapeutic agent for delivery to a specific target. The target can be any desired intracellular target. In some embodiments, the target is a nucleic acid sequence or gene. In embodiments where it is desired to manipulate, modulate or edit a gene, the protein or peptide is a gene or genome editing agent. In some embodiments, the gene editing agents comprise: transcriptional activators, transcriptional repressors, transcription factors, enhancer modulating molecules, recombinases, nucleases, nucleic acid binding-proteins, nucleic acid binding-polynucleotides or oligonucleotides, DNA-binding proteins or DNA-binding nucleic acids, or combinations thereof. In some embodiments, the target is a protein or peptide. Accordingly, in some embodiments, the chimeric or anionic molecule comprises one or more gene editing agents, transcriptional modulators, translational modulators, post-translational modulators, and/or modulators that regulate protein expression, function, activity or combinations thereof.

In other embodiments, the chimeric molecule further comprises one or more detectable labels, anions, radiolabels, tags, targeting agents, negatively charged proteins or peptides, or combinations thereof. These molecules can be selected based on the user's desired goal, e.g. for diagnostic or research purposes, or to increase the anionic charge, targeting signals and the like. Accordingly, a liposomal formulation for complexing protein and nucleic acid (e.g. transcription factors with their target binding region as oligonucleotides) for inner ear cell types delivery in vivo, is used to treat deafness or associated disorders thereof as the chimeric molecule can be tailored for regeneration (e.g. hair cell and auditory neuron regeneration), repair (e.g. re-establishment of connections between hair cells and neurons for hearing recovery) and prevention.

In other embodiments, a chimeric molecule comprises at least one protein or peptide fused, complexed or linked to one or more anionic molecules. Preferably, the one or more anionic molecules comprise one or more anionic domains or bind to an anionic nucleic acid domain. In embodiments, the chimeric molecule comprises an overall net negative charge. In some embodiments, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. In some embodiments, the oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), a short interfering RNA (siRNA), a micro, interfering RNA (miRNA), a small, temporal RNA (stRNA), a short, hairpin RNA (shRNA), mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof. The chimeric molecule also comprises one or more proteins or peptides which are cationic, anionic or are neutrally charged. Examples of proteins include without limitation: enzymes, hormones, chemotherapeutic agents, immunotherapeutic agents, genome or gene editing agents, synthetic molecules or combinations thereof. The gene or genome editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In other embodiments, the chimeric molecule optionally comprises one or more detectable labels, radiolabels, tags, anions, targeting agents or combinations thereof.

In other embodiments, a cationic liposome encapsulates an anionic molecule comprising a protein or peptide complexed, fused or linked to a negatively charged molecule. In some embodiments, the negatively charged molecule comprises oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. In other embodiments, the polynucleotide or oligonucleotide is a guide RNA. In some embodiments, the protein or peptide is a negatively charged protein. In yet other embodiments, the one or more proteins or peptides are cationic, anionic or are neutrally charged. In yet another embodiment, the negatively charged protein is fused or linked to one or more proteins or peptides. In some embodiments, the protein or peptide comprises: enzymes, hormones, chemotherapeutic agents, immunotherapeutic agents, gene editing agents, synthetic molecules or combinations thereof. In some embodiments, the gene editing agents comprise: transcriptional activators, transcriptional repressors, transcription factors, enhancer modulating molecules, recombinases, nucleases, nucleic acid binding-proteins, nucleic acid binding-polynucleotides or oligonucleotides, DNA-binding proteins or DNA-binding nucleic acids, or combinations thereof. Examples of these gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof. The anionic molecule optionally comprises one or more detectable labels, radiolabels, tags, negatively charged proteins or peptides, anions, targeting agents or combinations thereof.

In some embodiments, a molecule comprises any one or more sequences comprising SEQ ID NOS: 1 to 95. In another embodiment, a molecule comprises any one or more sequences comprising SEQ ID NOS: 1 to 95, wherein each sequence further comprises one or more antimicrobial or membrane destabilizing domains, for example, aurein or fragments thereof.

In other embodiments, the liposome comprises one or more cationic lipids, modified lipids or combinations thereof.

In some embodiments, a liposome encapsulating one or more molecules embodied herein comprises a liposome, a nanoliposome, a niosome, a microsphere, a nanosphere, a nanoparticle, a micelle, or an archaeosome.

Modified Proteins or Peptides:

Hybrid proteins comprising a polypeptide or fragment thereof may be linked to other types of polypeptides, for example, a negatively supercharged protein, e.g. green fluorescent protein in addition to a reporter polypeptide, or in lieu of a reporter polypeptide. These additional polypeptides may be any amino acid sequence useful for the purification, identification, overall charge of the protein or peptide, and/or therapeutic or prophylactic application of the peptide. In addition, the additional polypeptide can be a signal peptide, or targeting peptide, etc.

In some cases, the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the polypeptide or increase affinity of the polypeptide for its appropriate receptor, ligand and/or binding proteins. In some cases, the other additions, substitutions or deletions may increase the solubility of the polypeptide. In some embodiments sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid for the purpose of increasing the polypeptide solubility following expression in recombinant host cells. In some embodiments, the polypeptides comprise another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, modulates (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bio-availability, facilitates purification, or improves or alters a particular route of administration. Similarly, the non-natural amino acid polypeptide can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification, transport through tissues or cell membranes, prodrug release or activation, size reduction, or other traits of the polypeptide.

The methods and compositions described herein include incorporation of one or more non-natural amino acids into a polypeptide. One or more non-natural amino acids may be incorporated at one or more particular positions which does not disrupt activity of the polypeptide. This can be achieved by making “conservative” substitutions, including but not limited to, substituting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, bulky amino acids with non-natural or natural bulky amino acids, hydrophilic amino acids with non-natural or natural hydrophilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.

A variety of biochemical and structural approaches can be employed to select the desired sites for substitution with a non-natural amino acid within the polypeptide. Any position of the polypeptide chain is suitable for selection to incorporate a non-natural amino acid, and selection may be based on rational design or by random selection for any or no particular desired purpose. Selection of desired sites may be based on producing a non-natural amino acid polypeptide (which may be further modified or remain unmodified) having any desired property or activity, including but not limited to agonists, super-agonists, partial agonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, modulators of binding to binder partners, binding partner activity modulators, binding partner conformation modulators, dimer or multimer formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability. For example, locations in the polypeptide required for biological activity of a polypeptide can be identified using methods including, but not limited to, point mutation analysis, alanine scanning or homolog scanning methods. Residues other than those identified as critical to biological activity by methods including, but not limited to, alanine or homolog scanning mutagenesis may be good candidates for substitution with a non-natural amino acid depending on the desired activity sought for the polypeptide. Alternatively, the sites identified as critical to biological activity may also be good candidates for substitution with a non-natural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to make serial substitutions in each position on the polypeptide chain with a non-natural amino acid and observe the effect on the activities of the polypeptide. Any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described herein.

Modified Oligonucleotides:

Examples of some oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, modified oligonucleotides comprise those with phosphorothioate backbones and those with heteroatom backbones, CH₂—NH—O—CH₂, CH, —N(CH₃)—O—CH₂ [known as a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH,). The amide backbones disclosed by De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374) are also embodied herein. In some embodiments, the oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506), peptide nucleic acid (PNA) backbone wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497). Oligonucleotides may also comprise one or more substituted sugar moieties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N₆ (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al. Ann. N.Y. Acad. Sci. 1992, 660, 306; Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327; Svinarchuk et al. Biochimie 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). Oligonucleotides comprising lipophilic moieties and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.

Labeled Molecules:

In another preferred embodiment, the chimeric molecules can be labeled. Uses include therapeutic and imaging for diagnostic and prognostic purposes. The label may be a radioactive atom, an enzyme, or a chromophore moiety. Methods for labeling antibodies have been described, for example, by Hunter and Greenwood, Nature, 144:945 (1962) and by David et al. Biochemistry 13:1014-1021 (1974). Additional methods for labeling antibodies have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090. Methods for labeling oligonucleotide probes have been described, for example, by Leary et al. Proc. Natl. Acad. Sci. USA (1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435; Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al. Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem. (1984) 138:267.

The label may be radioactive. Some examples of useful radioactive labels include ³²P, ¹²⁵I, ¹³¹I, and ³H. Use of radioactive labels has been described in U.K. 2,034,323, U.S. Pat. No. 4,358,535, and U.S. Pat. No. 4,302,204.

Some examples of non-radioactive labels include enzymes, chromophores, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), β-galactosidase (fluorescein β-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels has been described in U.K. 2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci. USA, 47, 1981-1991 (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific chromophores useful in the present invention include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

The labels may be conjugated to the chimeric molecule by methods that are well known in the art. The labels may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate. Alternatively, labels such as enzymes and chromophores may be conjugated to the antibodies or nucleotides by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like.

In another preferred embodiment, the chimeric fusion molecules of the invention can be used for imaging. In imaging uses, the complexes are labeled so that they can be detected outside the body. Typical labels are radioisotopes, usually ones with short half-lives. The usual imaging radioisotopes, such as ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)TC, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹⁸F, ³H, ¹⁴C, ³⁵S or ³²P can be used. Nuclear magnetic resonance (NMR) imaging enhancers, such as gadolinium-153, can also be used to label the complex for detection by NMR. Methods and reagents for performing the labeling, either in the polynucleotide or in the protein moiety, are considered known in the art.

Reporter genes useful in the present invention include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.

Methods of Treatment

The compositions and molecules embodied herein are useful in those diseases and conditions that would benefit from protein therapeutics. In some embodiments, a method of treatment comprises administering to a patient an effective amount of cationic liposome encapsulating a chimeric molecule embodied herein. In other embodiments, the molecule comprises one or more sequences set forth as SEQ ID NOS: 1 to 32.

In another embodiment, a method of treatment comprises administering to a patient a therapeutically effective amount of a chimeric molecule comprising any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide, antimicrobial or membrane destabilizing peptide, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a method of treatment comprises administering to a patient a therapeutically effective amount of a chimeric molecule comprising any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide and an antimicrobial or membrane destabilizing peptide and one or more transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a method of treatment comprises administering to a patient a therapeutically effective amount of a chimeric molecule comprising any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: an antimicrobial or membrane destabilizing domain and one or more supercharged proteins or peptides, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a method of treatment comprises administering to a patient a therapeutically effective amount of a chimeric molecule comprising an antimicrobial or membrane destabilizing domain, one or more supercharged proteins or peptides, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In another embodiment, a method of treatment comprises administering to a patient a therapeutically effective amount of a chimeric nucleic acid molecule encoding any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide, antimicrobial or membrane destabilizing domain, transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof. In embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In other embodiments, the method of treating hearing loss or deafness using the liposomal formulation for complexing protein and nucleic acid embodied herein (e.g. transcription factors with their target binding region as oligonucleotides) for inner ear cell type delivery in vivo, comprises regeneration (e.g. hair cell and auditory neuron regeneration), repair (e.g. re-establishment of connections between hair cells and neurons for hearing recovery) and prevention.

Hearing Loss or Deafness and Associated Disorders:

One in 1000 newborns suffers from genetic deafness. Over 80 deafness genes have been identified, and additional 200-300 deafness genes remain to be discovered. Despite the tremendous progress, there is no treatment for any genetic deafness. Thus there are urgent needs to develop treatment that targets different types of genetic deafness.

There two main categories of genetic deafness: recessive deafness that is generally congenital; and dominant deafness that is mainly progressive. For recessive deafness, delivery and continuous expression of a normal copy of mutant gene could compensate for lost function for hearing recovery. Adeno-associated virus (AAV) based gene therapy has been the choice to be developed as treatment for recessive deafness, due to its long-term expression pattern and good safety record. AAV vectors however can only accommodate inserts less than 4.5 kb, whereas many deafness genes are much larger in size, thus severely limiting usefulness of AAV. For dominant deafness gene delivery will unlikely work.

Non-inherited abnormalities of the inner ear, such as the Mondini malformation, account for roughly 20% of congenital sensorineural deafness. The bulk of the remaining (genetic) deafness is non-syndromic, meaning that it does not have any obvious distinguishing features.

Most non-syndromic hearing losses are caused by connexin gene mutations. In the mammals, at least 20 connexin subtypes have been identified in mouse and human genomics. Connexin genes encode gap junctional channels, which connect two adjacent cells allowing passage of cytoplasmic ions and small molecules up to 1.2 kDa. In the mammalian inner ear, connexin26 (Cx26) and Cx30 are predominant isoforms. Cx26 mutation can induce a high incidence of hearing loss, responsible for 70 to 80 percent of nonsyndromic hearing loss in children.

Non-Syndromic Deafness:

Nonsyndromic means that deafness occurs in isolation, without other associated disorders. About 80% of genetic hearing loss is non-syndromic. Between 1992 and 2001, 38 loci for autosomal dominant non-syndromic deafness have been mapped and 11 genes have been identified. Autosomal dominant loci are called DFNA, autosomal recessive as DFNB, and X-linked as DFN.

Non-syndromic deafness is highly heterogeneous, but mutations in the connexin-26 molecule (gap junction protein, gene GJB2) account for about 49% of patients with non-syndromic deafness and about 37% of sporadic cases. About 1 in 31 individuals of European extraction are likely carriers.

Autosomal Dominant (DFNA):

Autosomal dominant deafness is passed directly through generations. It is often possible to identify an autosomal dominant pattern through simple inspection of the family tree. Examples of autosomal dominant deafness are missense mutation in COL11A2 (DFNA13) and in the TMC1 gene. COL11A2 encodes a chain of type XI collagen whereas TMC1 encodes a hair cell channel protein.

Autosomal Recessive (DFNB):

Autosomal recessive disorders require a gene from both the mother and father.

Syndromic Deafness:

Syndromic deafness, which accounts for the remaining 20% of congenital deafness, comprises an immensely complicated interlinked set of disorders. The descriptions here are only to give the general flavor of the diseases and are not meant to include all features of the disorders. In most cases, an Online Mendelian Inheritance in Man (OMIM) database link to the main type of the genetic disorder is provided. This database is a catalog of human genes and genetic disorders.

Alport Syndrome:

Alport syndrome is caused by mutations in COL4A3, COL4A4 or COL4A5. The classic phenotype is renal failure and progressive sensorineural deafness.

Branchio-Oto-Renal Syndrome:

Branchio-oto-renal syndrome is caused by mutations in EYA1, a gene of 16 exons within a genomic interval of 156 kB. This syndrome is characterized by hearing disturbances and cataract, branchial cleft fistulae, and preauricular pits. Mondini malformations and related dysplasias may occur.

X-Linked Charcot Marie Tooth (CMT):

The dominantly form of X-linked CMT is caused by a mutation in the connexin 32 gene mapped to the Xq13 locus. Usual clinical signs consist of a peripheral neuropathy combined with foot problems and “champagne bottle” calves.

As noted above, the connexin gene is also associated with a large percentage of cases of non-syndromic deafness. There are several other associated neuropathies and deafness syndromes. Autosomal recessive demyelinating neuropathy, autosomal dominant hereditary neuropathies type I and II, and X-linked hereditary axonal neuropathies with mental retardation are all associated with deafness.

Goldenhar's Syndrome:

Oculoauriculovertebral dysplasia (OAVD) or Goldenhar's syndrome was originally described in 1881. It includes a complex of features including hemifacial microtia, otomandibular dysostosis, epibulbar lipodermoids, coloboma, and vertebral anomalies that stem from developmental vascular and genetic field aberrations. It has diverse etiologies and is not attributed to a single genetic locus. The incidence is roughly 1 in 45,000.

Jervell and Lange-Nielsen Syndrome:

Jervell and Lange-Nielsen Syndrome is associated with cardiac arrhythmias. There is, by prolongation of the QT interval, torsade de Pointe arrhythmias (turning of the points, in reference to the apparent alternating positive and negative QRS complexes), sudden syncopal episodes, and severe to profound sensorineural hearing loss.

Mohr-Tranebjaerg Syndrome (DFN-1):

Mohr-Tranebjaerg syndrome (DFN-1) is an X-linked recessive syndromic hearing loss characterized by postlingual sensorineural deafness in childhood, followed by progressive dystonia, spasticity, dysphagia and optic atrophy. The syndrome is caused by a mutation thought to result in mitochondrial dysfunction. It resembles a spinocerebellar degeneration called Fredreich's ataxia which also may exhibit sensorineural hearing loss, ataxia and optic atrophy. The cardiomyopathy characteristic of Freidreich's ataxia is not seen in Mohr-Tranebjaerg syndrome.

Norrie Disease:

Classic features of Norrie Disease include specific ocular symptoms (pseudotumor of the retina, retinal hyperplasia, hypoplasia and necrosis of the inner layer of the retina, cataracts, phthisis bulbi), progressive sensorineural hearing loss, and mental disturbance, although less than one-half of patients are hearing impaired or mentally retarded.

Pendred Syndrome:

Pendred Syndrome is deafness associated with thyroid disease (goiter).

Stickler Syndrome:

Stickler syndrome is caused by mutations in COL11. It is characterized by hearing impairment, midface hypoplasia, progressive myopia in the first year of life, and arthropathy.

Treacher Collins Syndrome:

Treacher Collins syndrome (OMIM entry TCOF1) is characterized by coloboma of the lower eyelid (the upper eyelid is involved in Goldenhar syndrome), micrognathia, microtia, hypoplasia of the zygomatic arches, macrostomia, and inferior displacement of the lateral canthi with respect to the medial canthi.

Waardenburg Syndrome:

The clinical symptoms of Waardenburg Syndrome (WS) type I and II include lateral displacement of the inner canthus of each eye, pigmentary abnormalities of hair, iris, and skin (often white forelock and heterochromia iridis), and sensorineural deafness. The combination of WS type I characteristics with upper limb abnormalities has been called Klein-Waardenburg syndrome or WS type III. The combination of recessively inherited WS type II characteristics with Hirschsprung disease has been called Waardenburg-Shah syndrome or WS type IV.

Usher Syndrome:

Usher syndrome is characterized by hearing impairment and retinitis pigmentosa. Usher syndrome can be classified into three different types on the basis of clinical findings. In type I, there is both hearing impairment and vestibular impairment. In type II, there is hearing impairment without vestibular impairment. In type III, there are variable amounts of vestibular impairment.

Mitochondrial Disorders:

Hearing loss is common in mitochondrial disorders including MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke like episodes), Kearns-Sayre syndrome and MERRF (myoclonic epilepsy with ragged red fibers). These disorders are caused by mutations in mitochondrial DNA, and are characterized by muscular weakness, an abnormal muscle biopsy with “ragged red” fibers, and a variety of other findings that define the specific clinical phenotype. In MELAS, it was reported that the hearing loss is caused by cochlear damage. It resembles presbyacusis in that it is generally symmetrical, gradual, and affects the higher frequencies first. Others have also reported hearing loss associated with mitochondrial mutations. Mitochondrial DNA mutations accumulate naturally during life and are presently implicated as an important cause of normal aging. Mitochondrial defects have been reported to cause both unusual sensitivity to aminoglycosides as well as non-syndromic sensorineural deafness.

Non-Inherited Congenital Deafness:

These types of abnormalities account for roughly 20% of congenital deafness, the remainder being genetic in origin.

Mondini Dysplasia:

The normal cochlea has two and one-half turns. A cochlear malformation consists of a membranous abnormality, a bony abnormality, or a combination of these two. If cochlear development is arrested in the embryo, a common cavity may occur instead of the snail like cochlea. This is called the Mondini dysplasia or malformation.

Often accompanying the Mondini dysplasia is abnormal communication between the endolymphatic and perilymphatic spaces of the inner ear and subarachnoid space. It is usually caused by a defect in the cribiform area of the lateral end of the internal auditory canal, presumably because of this abnormal channel, perilymphatic fistulae are more common in this disorder.

A related anomaly and more severe syndrome, the CHARGE association, consists of coloboma, heart disease, choanal atresia, retarded development, genital hypoplasia, ear anomalies including hypoplasia of the external ear and hearing loss. These individuals have a Mondini type deformity and absence of semicircular canals.

Enlarged Vestibular Aqueduct Syndrome:

Enlarged Vestibular Aqueduct Syndrome is defined on the CT scan as a diameter greater than or equal to 1.5 mm measured midway between the operculum and the common crus.

Recently CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) endonuclease gene editing has been developed with potential to revolutionize genetic therapy. CRISPR uses Cas9 and guide RNA to target any genomic sequence for specific cleavage, resulting disruption or repair of any gene. The process applies to mutant genes regardless the nature of mutations (recessive or dominant), with permanent correction to restore normal gene function.

Conventional approaches with CRSIP involve the use of viral vehicle to deliver Cas9 and guide RNAs (sgRNA, a template homologous to the target genomic region of 20-29 bp) to cells for gene editing. However the viral genome will remain permanently inside cells (for inner ear it means the whole life) with uncertain consequences (e.g. immunogenic response, potential recombination). In addition the efficiency of CRISPR mediated targeted cleavage in vivo has been relatively low (less than 5%).

A major improvement over previous methods is to directly deliver protein and nucleic acid complex into cells for the CRISPR mediated gene editing. This approach would allow transient delivery of proteins and nucleic acids, which will be degraded after their function, thus limiting possible adverse effect due to long-term presence of both in cells. Delivery of the combination of proteins with nucleic acids has not been achieved in vivo or in vitro.

Nucleic acid deliveries based on cationic lipid formulations have been used widely with high efficiency. The lipid bilayer of liposome protects the encapsulated nucleic acids from degradation and can prevent neutralization by antibodies. Significantly, fusion of liposomes with the endosomal membrane during endosome maturation can enable the efficient endosomal escape of cationic lipid-delivered cargo. As some natural proteins or proteins with modifications can be highly negative (anionic), it is possible to use liposomes based vehicles to deliver proteins into cells directly with high efficiency. It is further possible to combine the delivery of anionic proteins and nucleic acids (which is anionic) together with liposomes.

Accordingly, in some embodiments, a method of treating deafness associated with a genetic mutation in a patient in need thereof, comprises administering to the patient a therapeutically effective amount of a chimeric molecule comprising at least one protein or peptide fused, complexed or linked to one or more anionic molecules. The chimeric molecule targets one or more genetic loci associated with deafness in a patient and modulates replication, expression, function or activity of the genetic locus. The genotypic variations that can confer abnormal phenotypes, e.g. deafness, comprise: mutations, insertions, deletions, substitutions or combinations thereof wherein the abnormal gene is expressed. In embodiments, the chimeric molecule comprises one or more gene editing agents for repression of the genetic locus associated with deafness in a patient. These gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.

In embodiments, the anionic molecules comprise: oligonucleotides, polynucleotides, proteins, peptides, peptide nucleic acids (PNA), synthetic molecules or combinations thereof. Examples of oligonucleotides or polynucleotides include: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof.

In embodiments, the chimeric molecule is encapsulated in a cationic liposome and is administered to a patient's inner ear.

In another embodiment, a method of treating a patient suffering from deafness due to a genetic mutation comprises: administering to a patient's inner ear, a cationic liposome encapsulating a therapeutically effective amount of an anionic molecule comprising a protein or peptide complexed, fused or linked to a negatively charged molecule. In these embodiments, the chimeric molecule targets one or more genetic loci associated with deafness in a patient and modulates replication, expression, function or activity of the genetic locus. These genetic loci associated with deafness comprise: mutations, insertions, deletions, substitutions or combinations thereof. The anionic molecule comprises one or more gene editing agents for repression of a genetic locus associated with deafness in a patient. Examples of these gene editing agents comprise: transcriptional activators, transcriptional repressors, transcription factors, enhancer modulating molecules, recombinases, nucleases, nucleic acid binding-proteins, nucleic acid binding-polynucleotides or oligonucleotides, DNA-binding proteins or DNA-binding nucleic acids, or combinations thereof.

Non-exhaustive examples of mutations in genes that cause, for example, nonsyndromic deafness, include, without limitation, mutations in the ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, ESPN, EYA4, GJB2, GJB6, GRXCR1, KCNQ4, MYO3A, MYO15A, MYO6, MYO7A, OTOF, OTOA, PCDH15, POU3F4, RDX, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, USH1C, WFS1 and WHRN genes cause nonsyndromic deafness, with weaker evidence currently implicating genes CCDC50, DIAPH1, DSPP, ESRRB, GJB3, GRHL2, GRXCR1, HGF, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR96, MYH14, MYH9, MYO1A, MYO3A, OTOA, PJVK, POU4F3, PRPS1, PTPRQ, RDX, SERPINB6, SIX1, SLC17A8, TPRN, TRIOBP, and WHRN.

Accordingly, any one or more genes or genetic loci associated with deafness can be targeted. In other embodiments, the molecules embodied herein are administered to treat patients suffering from diseases or disorders associated with deafness. Examples of these diseases or disorders include: tinnitus, hyperscusis, ADHD.

In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.

In other embodiments, the anionic molecule comprises any one or more sequences having a sequence identity of at least about 75% to sequences set forth as SEQ ID NOS: 1 to 95. In another embodiment, the anionic molecule comprises one or more antimicrobial or membrane destabilizing domains.

In another embodiment, the one or more sequences are set forth as SEQ ID NOS: 1 to 95. In another embodiment, the one or more sequences comprise SEQ ID NOS: 1 to 95 linked, fused, complexed with one or more antimicrobial or membrane destabilizing domains.

In other embodiments, the chimeric molecules or the encapsulated chimeric or anionic molecules are administered in a pharmaceutical composition.

Pharmaceutical Compositions:

The types and amounts of chimeric molecules for use as therapeutic compounds may be believed to have therapeutic activity on the basis of any information available to the artisan. For example, a prototype compound may be believed to have therapeutic activity on the basis of information contained in the Physician's Desk Reference. In addition, by way of non-limiting example, a therapeutic compound may be believed to have therapeutic activity on the basis of experience of a clinician, structure of the compound, structural activity relationship data, EC₅₀, assay data, IC₅₀ assay data, animal or clinical studies, or any other basis, or combination of such bases.

A therapeutically-active compound is a compound that has therapeutic activity, including for example, the ability of a compound to induce a specified response when administered to a subject or tested in vitro. Therapeutic activity includes treatment of a disease or condition, including both prophylactic and ameliorative treatment. Treatment of a disease or condition can include improvement of a disease or condition by any amount, including prevention, amelioration, and elimination of the disease or condition. Therapeutic activity may be conducted against any disease or condition, including in a preferred embodiment against any disease or disorder that would benefit from dissociation of a tissue or mass of cells, for example. In order to determine therapeutic activity any method by which therapeutic activity of a compound may be evaluated can be used. For example, both in vivo and in vitro methods can be used, including for example, clinical evaluation, EC₅₀, and IC₅₀ assays, and dose response curves.

In some embodiments, a pharmaceutical composition comprises a cationic lipid encapsulating a chimeric molecule embodied herein. In other embodiments, the molecule comprises one or more sequences set forth as SEQ ID NOS: 1 to 95. In another embodiment, the one or more sequences comprise SEQ ID NOS: 1 to 95 linked, fused, complexed with one or more antimicrobial or membrane destabilizing domains.

Formulations, Administration:

The compositions embodied herein, are formulated for administration by any suitable method, for example, as described in Remington: The Science And Practice Of Pharmacy (21st ed., Lippincott Williams & Wilkins). Exemplary routes of administration include, but are not limited to parenteral, oral, subcutaneous, topical, intramuscular, transdermal, transmucosal, sublingual, intranasal, transvascular, subcutaneous, orbital, or combinations thereof.

Kits: In yet another aspect, the invention provides kits for targeting nucleic acid sequences of cells and molecules associated with modulation of the target molecule. For example, the kits can be used to target any desired nucleic sequence and as such, have many applications.

In one embodiment, a kit comprises: (a) a cationic lipid, and a chimeric molecule or an encapsulated chimeric molecule, or a protein and a separate polyanionic molecule, or any combinations thereof, and (b) instructions to administer to cells or an individual a therapeutically effective amount of the composition. In some embodiments, the kit may comprise pharmaceutically acceptable salts or solutions for administering the composition. Optionally, the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert. For example, the kit may have standard instructions informing a physician or laboratory technician to prepare a dose of chimeric molecule.

Optionally, the kit may further comprise a standard or control information so that a patient sample can be compared with the control information standard to determine if the test amount of chimeric molecule is a therapeutic amount consistent with for example, treating deafness in a patient.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, applicants do not admit any particular reference is “prior art” to their invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

Examples

The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example 1: Efficient Delivery of Genome Editing Proteins In Vitro and In Vivo

Construction of Cas9, Cre, and TALE Fusion and sgRNA Expression Plasmids.

Sequences of all constructs used are listed below. All protein constructs were generated from previously reported plasmids for protein of interest cloned into a pET29a expression plasmid.

Expression and Purification of S. pyogenes Cas9 and Other Proteins.

E. coli BL21 STAR (DE3) competent cells (Life Technologies) were transformed with pMJ806 (Pattanayak, V. et al. Nat. Biotechnol. 31, 839-843 (2013)) encoding the S. pyogenes Cas9 fused to an N-terminal 10×His-tag/maltose binding protein. The resulting expression strain was inoculated in Luria-Bertani (LB) broth containing 100 μg/mL of ampicillin at 37° C. overnight. The cells were diluted 1:100 into the same growth medium and grown at 37° C. to OD₆₀₀=˜0.6. The culture was incubated at 20° C. for 30 min, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce Cas9 expression. After ˜16 h, the cells were collected by centrifugation at 8,000 g and resuspended in lysis buffer (50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 8.0, 1 M NaCl, 20% glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP)). The cells were lysed by sonication (1 sec pulse-on, 1 sec pulse-off for 15 min total at 6 W output) and the soluble lysate was obtained by centrifugation at 20,000 g for 30 min.

The cell lysate was incubated with His-Pur nickel-nitriloacetic acid (nickel-NTA) resin (Thermo Scientific) at 4° C. for 30 min to capture His-tagged Cas9. The resin was transferred to a 20-mL column and washed with 20 column volumes of lysis buffer. Cas9 was eluted in 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, 10 mM TCEP, and 300 mM imidazole, and concentrated by Amicon ultra centrifugal filter (Millipore, 100-kDa molecular weight cut-off) to ˜50 mg/mL. The 6×His tag and maltose-binding protein were removed by TEV protease treatment at 4° C. for 20 hours and captured by a second Ni-affinity purification step. The eluent, containing Cas9, was injected into a HiTrap SP HP column (GE Healthcare) in purification buffer containing 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, and 10 mM TCEP. Cas9 was eluted with purification buffer containing a linear NaCl gradient from 0.1 M to 1 M over five column volumes. The eluted fractions containing Cas9 were concentrated down to a concentration of 200 μM as quantified by bicinchoninic acid assay (BCA) (Pierce Biotechnology), snap-frozen in liquid nitrogen, and stored in aliquots at −80° C. All other proteins were purified by this method but without TEV cleavage step and proteins containing (−30) GFP were purified by anion exchange using a Hi-Trap Q HP anion exchange column (GE Healthcare) using the same purification protocol.

In Vitro Transcription of sgRNAs.

Linear DNA fragments containing the T7 promoter binding site followed by the 20-bp sgRNA target sequence were transcribed in vitro using the T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's instructions. In vitro transcribed RNA was precipitated with ethanol and purified by gel electrophoresis on a Criterion 10% polyacrylamide TBE-Urea gel (Bio-Rad). Excised gel fragments were extracted in 420 μL of 300 mM NaCl overnight on a rocking surface at 4° C. Gel-purified sgRNA was precipitated with ethanol and redissolved in water and sgRNA concentration was finally quantified by UV absorbance and snap-frozen at −80° C.

Plasmid Transfection.

Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies) according the manufacturer's protocol. For TALE activator plasmids, 300 ng of DNA was transfected, and for the activator synergy experiments 60 ng of each of five plasmids was pooled and transfected. For Cas9 nuclease delivery experiments, linear DNA PCR products expressing sgRNAs were used in transfection experiments targeting genomic sites in CLTA, EMX, VEGF, and GFP (sgRNA GFP g1, GFP g3, GFP g5, and GFP g7 for nickase studies). Linear DNA PCR products were generated using plasmid containing the U6 promoter as template and forward primers bearing the U6 promoter upstream sequence and reverse primers containing U6 downstream sequence followed by the sgRNA sequence (20-bp sequence unique to each target plus constant sgRNA backbone architecture sequence). sgRNAs expressed from linear DNA templates contained at least two 5′ guanosines to match in vitro transcribed sgRNAs that required these bases for T7 transcription. Primer sequences and PCR conditions are referred to herein. For dCas9 activator experiments, 700 ng of Cas9 or dCas9-VP64 plasmid DNA was co-transfected with 250 ng of the appropriate sgRNA expression plasmid. For activator synergy experiments 50 ng of DNA from each of the six sgRNA was pooled and co-transfected with 700 ng of dCas9-VP64 plasmid.

Delivery of Transcription Factor Proteins Complexed with Cationic Lipids in Cell Culture.

Briefly, cultured cells were plated in 48-well format (250 μL volume) in Dulbecco's Modified Eagle's Media plus GLUTAMAX (Life Technologies, Carlsbad, Calif.) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ˜70% confluence the next day. Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery. Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 μM protein (in 275 μL final volume) with 0.5-1.5 μL of commercially available cationic lipids in 25 μL OPTIMEM media (Life Technologies, Carlsbad, Calif.) according to the manufacturer's protocol for normal plasmid transfection, including incubation time. For Cas9 delivery in vitro, transcribed sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent. 25 μL lipid complexes in OPTIMEM media were added to cells and media was replaced 12-16 hours later fresh media unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for gene activation either 4 or 16 hours after delivery, and for gene modification 48 hours after delivery.

T7 Endonuclease I Assay to Detect Genomic Modifications.

U2OS-EGFP cells or HEK293T cells were transfected with Cas9 expression and sgRNA expression plasmids or linear DNA PCR products as described above or treated with only Cas9 protein, only in vitro transcribed sgRNA, or only RNAiMAX. Genomic DNA was isolated from cells 2 days after transfection using the DNAdvance Kit (Agencourt) following the manufacturer's instructions. 200 ng of genomic DNA was used as template in PCR reactions to amplify the targeted genomic loci with flanking survey primer pairs specified herein. PCR products were purified with a QlAquick PCR Purification Kit (Qiagen) and quantified with QUANT-IT™ PICOGREEN® dsDNA Kit (Life Technologies). 250 ng of purified PCR DNA was combined with 2 μL of NEBuffer 2 (NEB) in a total volume of 19 μL and denatured then re-annealed with thermocycling at 95° C. for 5 min, 95 to 85° C. at 2° C./s; 85 to 20° C. at 0.2° C./s. The re-annealed DNA was incubated with 1 μl of T7 Endonuclease I (10 U/μ1, NEB) at 37° C. for 15 min. 10 μL of 50% glycerol was added to the T7 Endonuclease reaction and 12 μL was analyzed on a 5% TBE 18-well Criterion PAGE gel (Bio-Rad) electrophoresed for 30 min at 200 V, then stained with 1×SYBR Gold (Life Technologies) for 30 min. Cas9-induced cleavage bands and the uncleaved band were visualized on an Alphalmager HP (Alpha Innotech) and quantified using ImageJ software (Schneider, C. A., et al. Nat. Methods 9, 671-675 (2012)). The peak intensities of the cleaved bands were divided by the total intensity of all bands (uncleaved+cleaved bands) to determine the fraction cleaved which was used to estimate gene modification levels as previously described (Guilinger, J. P., et al., Nat. Biotechnol. 32, 577-582 (2014)). For each sample, transfections and subsequent modification measurements were performed in triplicate on different days.

Stem Cell Culture and Delivery.

Mouse embryonic stem cell (ES) line Tau-GFP (courtesy of Dr. A. Edge, Massachusetts Eye & Ear Infirmary, Boston) containing a permanent GFP gene insertion was cultured in DMEM with 10% FBS (Gibco), 100 mM MEM nonessential amino acids (Gibco), 0.55 mM 2-mercaptoethanol, and leukemia inhibitory factor (1,000 units/ml; Chemicon). After 3 days floating spheres were formed that exhibited GFP fluorescence. Complexes of Cas9:sgRNA and Lipofectamine 2000 were added to the culture containing the floating spheres for 16 hours. After Cas9:sgRNA treatment, the cells were cultured in the above media for 3 days. The floating spheres were treated with trypsin for 5 min then passed through a 70 μm filter to collect single cells. The cells were cultured on laminin-coated slides in DMEM/F12 (1:1) supplemented with 1×N2, 1×B27, penicillin-streptomycin (100 μg/mL) and 10% FBS for two days before labeling. Immunohistochemistry was performed using an anti-GFP antibody (#ab13970, Abcam) to assess GFP expression. To quantify the number of GFP-negative cells, the total number of GFP-positive and GFP-negative cells from three representative visual fields at 20× magnification were counted, and the average efficiency was calculated. Three independent experiments were performed for each condition.

Microinjection of Proteins to Mouse Inner Ear.

P1 floxP-tdTomato mice (The Jackson Laboratory) were used for (−30)GFP-Cre injection and P2 Atoh1-GFP mice (Dr. J Johnson, Southwestern Medical Center, University of Texas) were used for Cas9:sgRNA injection. Animals were used under protocols approved by the Massachusetts Eye & Ear Infirmary ALCUC committee. Mice were anesthetized by lowering their temperature on ice. Cochleostomies were performed by making an incision behind the ear to expose the cochlea. Glass micropipettes held by a micromanipulator were used to deliver the complex into the scala media, which allows access to inner ear hair cells. For delivery of (−30)GFP-Cre, 3 μL of 45 μM protein was mixed with 3 μL of either RNAiMAX or Lipofectamine 2000 and incubated at room temperature for 30 minutes prior to injection. Four mice were injected per treatment group. For delivery of Cas9:sgRNA complexes, 1 μL of 200 μM Cas9 protein was mixed with 2 μL of 100 μM sgRNA and incubated for 5 minutes at room temperature before mixing with 3 μL of either RNAiMAX or Lipofectamine 2000 and incubating for an additional 30 minutes prior to injection. Three mice were injected per treatment group. The total delivery volume for every injection was 0.3 μL per cochlea and the release was controlled by a micromanipulator at the speed of 32 nL/sec.

Immunohistochemistry and Quantification.

5-10 days after injection, the mice were sacrificed and cochlea were harvested by standard protocols. For immunohistochemistry, antibodies against hair-cell markers (Myo7a and Esp) and supporting cells (Sox2) were used following a previously described protocol (Sage, C. et al. Science 307, 1114-1118 (2005)). To quantify the number of tdTomato positive cells after (−30)GFP-Cre or GFP negative cells after Cas9:sgRNA delivery, the total number of outer hair cells were counted in a region spanning 200 μm around the site of injection in the base turn of the cochlea. The efficiency of (−30)GFP-Cre-induced recombination or Cas9:sgRNA-induced genome modification was calculated as the percentage of outer hair cells that expressed tdTomato or that lost GFP expression.

High-Throughput DNA Sequencing of Genome Modifications.

HEK293T cells were either transfected with Cas9 and sgRNA expression plasmids or linear DNA PCR products or treated with 50 nM Cas9 protein, 250 nM purified sgRNA, and cationic lipids as described earlier for Cas9 protein delivery to U2OS-EGFP reporter cells. For plasmid-based transfection experiments, 700 ng of Cas9 expression plasmid plus 250 ng of sgRNA plasmid or 50 ng of a linear DNA PCR product expressing sgRNA for targeting either the EMX1, CLTA2, or VEGF locus were transfected with Lipofectamine 2000 (Life Technologies) and cells were isolated 2 days later. For protein delivery experiments in vivo, ˜30 mg of mouse tissue was isolated from anesthetized mice and genomic DNA was extracted using the Agencourt DNAAdvance Genomic DNA Isolation Kit (Beckman Coulter). For cell culture experiments genomic DNA was isolated as described above. 150 ng of genomic DNA was used as template to amplify by PCR the on-target and off-target genomic sites with flanking HTS primer pairs specified herein. Relative amounts of crude PCR products were quantified by gel electrophoresis and samples treated with different sgRNA pairs or Cas9 nuclease types were separately pooled in equimolar concentrations before purification with the QlAquick PCR Purification Kit (Qiagen). ˜150 ng of pooled DNA was electrophoresed using a 5% TBE 18-well Criterion PAGE gel (BioRad) for 30 min at 200 V and DNAs ˜125 bp to ˜300 bp in length were isolated and purified by QlAquick PCR Purification Kit (Qiagen). Purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced on a MiSeq high-throughput DNA sequencer (Illumina) as previously described (Pattanayak, V. et al. Nat. Biotechnol. 31, 839-843 (2013)).

Quantification of Cas9 Protein Uptake.

Alexa Fluor 647 C2 Maleimide (Life Technologies, Carlsbad Calif.) was used to fluorescently label Cas9 protein on surface cysteines. A 10 mM stock solution of Alexa647 was prepared in anhydrous DMSO. In a 0.4 mL reaction, 10 nmol of purified Cas9 protein and 200 nmol of Alexa647 maleimide were combined in buffer conditions used for Cas9 protein storage. The labeling reaction was incubated at 4° C. for 16 hours. At the end of the reaction, excess unconjugated Alexa647 was removed by re-purifying the labeled Cas9 protein by cation exchange chromatography as described above. To measure the amount of protein delivered into treated cells, 20,000 cells were plated in the wells of a 48-well plate 1 day prior to treatment. On the day of treatment, 50 nM of Alexa647-labeled Cas9 (Cas9-Alexa647) and 100 nM of EGFP1 sgRNA were prepared for delivery using 0.8 μL of Lipofectamine 2000 as described above, and applied to the cells. After 4 hours, Cas9-Alexa647:sgRNA Lipofectamine-containing media was removed, and cells were washed three times with 500 μL of PBS containing 20 U/mL heparin.

The cells were trypsinized and prepared for counting and flow cytometry as described above. Cas9-Alexa647 uptake was measured by flow cytometry, while 10,000 cells of the treated population were transferred to a black, flat-bottomed, opaque 96-well plate. Standard curves of Cas9-Alexa647 were prepared by complexing 50 pmol of the Cas9-Alexa647 protein with Lipofectamine 2000 exactly as described for Cas9-Alexa647 delivery, followed by serial 2-fold dilutions in DMEM with 10% FBS containing 10,000 U2OS cells per well in the 96-well plate. The effect of U2OS cells or complexation with Lipofectamine 2000 on Alexa647 fluorescence was determined by preparing three additional Cas9-Alexa647 standard curves: (i) with Lipofectamine 2000 in media lacking U2OS cells, (ii) without Lipofectamine 2000 in media containing U2OS cells, and (iii) without Lipofectamine 2000 in media lacking U2OS cells.

Data Analysis.

Illumina sequencing reads were filtered and parsed with scripts written in Unix Bash. DNA sequences will be deposited in NCBI's Sequencing Reads Archive (SRA) and source code can be found in Supplementary Software. Sample sizes for sequencing experiments were maximized (within practical experimental considerations) to ensure greatest power to detect effects. Statistical analyses for Cas9-modified genomic sites (Table 2) were performed as previously described (Sander, J. D. et al. Nucleic Acids Res. 41, e181 (2013)).

Amino acid sequences of proteins used in this study

(+36)GFP-Cre-6xHis (SEQ ID NO: 1): MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVP WPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKF EGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKD GSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHG RDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVR KNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQA RGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFE RTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGR TKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLS TRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGW TNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH (−7)GFP-Cre-6xHis (SEQ ID NO: 2): MGASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGM DELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRK NLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQAR GLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFER TDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRT KTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLST RALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWT NVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH (−20)GFP-Cre-6xHis (SEQ ID NO: 3): MGASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGS VQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGM DELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRK NLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQAR GLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFER TDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRT KTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLST RALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWT NVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH (−30)GFP-Cre-6xHis (SEQ ID NO: 4): MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPW PTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGS VQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGM DELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRK NLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQAR GLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFER TDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRT KTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLST RALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWT NVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH Cre-6xHis (SEQ ID NO: 5): MASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAW CKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAV SLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLL RIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVAD DPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSA RVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHH HHHH Cas9 (SEQ ID NO: 6): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9-6xHis (SEQ ID NO: 7): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHH HHHH NLS-Cas9-6xHis (SEQ ID NO: 8): MPKKKRKVMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEED KKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLI EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDHHHHHH Cas9-NLS-6xHis (SEQ ID NO: 9): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLyETRIDLSQLGGDPK KKRKVMDKHHHHHH (+36)dGFP-NLS-Cas9-6xHis (Y67S) (SEQ ID NO: 10): MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVP WPTLVTTLTSGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKF EGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKD GSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHG RDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGT NSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRY TRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFK SNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGG LSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHH (−30)dGFP-NLS-Cas9-6xHis (Y67S) (SEQ ID NO: 11): MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPW PTLVTTLTSGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEG DTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSV QLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMD ELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIK KGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELE NGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHHHHHH dCas9-VP64-6xHis (D10A and H840A) (SEQ ID NO: 12): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGS PKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAGGGGSGRADALDDFDLDMLGSDA LDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLHHHHHH Cas9 nickase (D10A) (SEQ ID NO: 13): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHH HHHH (−30)GFP-Cre-6xHis (SEQ ID NO: 14): ATGGGTGCTAGCAAAGGTGAAGAGCTGTTTGACGGTGTAGTACCGATCTTAGTGGA ATTAGACGGCGACGTGAACGGTCACGAATTTAGCGTGCGCGGCGAGGGCGAAGGTG ACGCTACCGAGGGTGAATTGACCCTGAAGTTTATTTGCACAACAGGCGAATTACCCG TTCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTGATTA CCCAGATCATATGGATCAACACGATTTTTTCAAATCAGCCATGCCTGAAGGATATGT TCAAGAGCGTACAATCAGCTTCAAGGACGATGGCACCTATAAAACGCGTGCGGAAG TGAAATTTGAAGGCGACACATTAGTAAACCGTATCGAACTGAAAGGTATCGACTTC AAAGAAGACGGCAACATTTTAGGCCATAAGCTGGAATATAACTTTAATTCTCATGAC GTGTATATTACGGCCGATAAACAGGAAAACGGTATCAAGGCAGAATTTGAAATTCG CCATAACGTGGAGGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGC CGATTGGTGATGGGCCTGTACTGTTACCTGACGATCACTACCTGAGCACGGAGTCAG CCCTGAGCAAAGATCCGAACGAAGACCGCGATCACATGGTTCTGTTAGAATTCGTG ACCGCTGCAGGCATTGATCATGGAATGGACGAGCTGTACAAGACCGGTGGTAGCGG TGGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGG CAGCGGCGGTACCGCGAGCAATTTACTGACCGTACACCAAAATTTGCCTGCATTGCC GGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATC GCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGG CGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTT CGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAA CATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGAC AGCAATGCTGTTTCACTGGTTATGCGGCGTATCCGAAAAGAAAACGTTGATGCCGGT GAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCA CTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATT GCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATC TCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGT TAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGC GATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGT CAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCT GGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGG TCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATA TGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAAT GTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGT GCGCCTGCTGGAAGATGGCGACGGCGGATCCCATCACCACCACCATCAC Cre-6xHis (SEQ ID NO: 15): ATGGCGAGCAATTTACTGACCGTACACCAAAATTTGCCTGCATTGCCGGTCGATGCA ACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTT TTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTG CAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCT TCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCA GCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGT TTCACTGGTTATGCGGCGTATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAA AACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAA ATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACA CCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTG ACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCA GGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTC CGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAA TGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGAT TTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATA CCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCG CTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTG TCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTG GAAGATGGCGACGGCGGATCCCATCACCACCACCATCAC (−30)dGFP-NLS-Cas9-6xHis (SEQ ID NO: 16): ATGGGTGCTAGCAAAGGTGAAGAGCTGTTTGACGGTGTAGTACCGATCTTAGTGGA ATTAGACGGCGACGTGAACGGTCACGAATTTAGCGTGCGCGGCGAGGGCGAAGGTG ACGCTACCGAGGGTGAATTGACCCTGAAGTTTATTTGCACAACAGGCGAATTACCCG TTCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTGATTA CCCAGATCATATGGATCAACACGATTTTTTCAAATCAGCCATGCCTGAAGGATATGT TCAAGAGCGTACAATCAGCTTCAAGGACGATGGCACCTATAAAACGCGTGCGGAAG TGAAATTTGAAGGCGACACATTAGTAAACCGTATCGAACTGAAAGGTATCGACTTC AAAGAAGACGGCAACATTTTAGGCCATAAGCTGGAATATAACTTTAATTCTCATGAC GTGTATATTACGGCCGATAAACAGGAAAACGGTATCAAGGCAGAATTTGAAATTCG CCATAACGTGGAGGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGC CGATTGGTGATGGGCCTGTACTGTTACCTGACGATCACTACCTGAGCACGGAGTCAG CCCTGAGCAAAGATCCGAACGAAGACCGCGATCACATGGTTCTGTTAGAATTCGTG ACCGCTGCAGGCATTGATCATGGAATGGACGAGCTGTACAAGACCGGTGGTAGCGG TGGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGG CAGCGGCGGTACCGCGCTCGCGCTGCCCAAGAAGAAGAGGAAGGTGATGGATAAG AAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCC ACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCG GAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCG TATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTT CTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCA TCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTAT CTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAAT CTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGAT TTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTAC AATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGAT TCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCC CGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGAC CCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAA AGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGC TGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA AGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTAC GATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCA GAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATT GATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAA AATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCA AGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGC ATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGA AGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGG CAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAA TTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGAC AAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTA TGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAAT GCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTT CAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAA TAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCAT TAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATG AAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATA GGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTG ATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTG ATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCA GATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTA AAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACAT ATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATT GAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGC GTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAG CATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAA AATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTAT GATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAG GTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAA GTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCAC TCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGA TAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGT GGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTAT TCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGA TTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGA GTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAG CAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTC TTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGA AACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAG TGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAG ACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGT AGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAA AATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAA AATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAAT CATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCT GGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATG TGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATA ACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTG AGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAG TTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAT ATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTG ATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTC TTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAG GAGGTGACCATCACCACCACCATCAC (+36)dGFP-NLS-Cas9 (SEQ ID NO: 17): ATGGGTGCTAGCAAAGGTGAACGTCTGTTTCGTGGTAAAGTACCGATCTTAGTGGAA TTAAAGGGCGACGTGAACGGTCATAAATTTAGCGTGCGCGGCAAAGGCAAAGGTGA CGCTACCCGTGGTAAATTGACCCTGAAGTTTATTTGCACAACAGGCAAATTACCCGT TCCGTGGCCCACCTTAGTGACCACCCTGACCTATGGCGTTCAGTGCTTCAGTCGTTA CCCTAAACATATGAAACGTCACGATTTTTTCAAATCAGCCATGCCTAAAGGATATGT TCAAGAGCGTACAATCAGCTTCAAGAAGGATGGCAAATATAAAACGCGTGCGGAAG TGAAATTTGAAGGCCGCACATTAGTAAATCGTATCAAACTGAAAGGTCGTGACTTCA AAGAAAAAGGCAACATTTTAGGCCATAAACTGCGTTATAACTTTAATTCTCATAAGG TGTATATTACGGCCGATAAACGCAAGAATGGTATCAAGGCAAAATTCAAAATTCGC CATAACGTGAAAGACGGCAGCGTTCAATTAGCGGATCATTATCAACAAAACACGCC GATTGGTCGCGGGCCTGTACTGTTACCTCGCAACCACTACCTGAGCACCCGTTCTAA ACTGAGCAAAGATCCGAAAGAAAAACGCGATCACATGGTTCTGTTAGAATTCGTGA CCGCTGCAGGCATTAAGCACGGACGCGACGAACGCTACAAGACCGGTGGTAGCGGT GGTTCTGGTGGTTCTGGTGGTAGCGGCGGTAGCGGTGGTAGCGGTGGTAGCGGTGG CAGCGGCGGTACCGCGCTCGCGCTGCCCAAGAAGAAGAGGAAGGTGATGGATAAG AAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCC ACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCG GAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCG TATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTT CTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCA TCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTAT CTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAAT CTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGAT TTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTAC AATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGAT TCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCC CGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGAC CCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAA AGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGC TGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA AGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTAC GATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCA GAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATT GATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAA AATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCA AGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGC ATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGA AGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGG CAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAA TTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGAC AAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTA TGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAAT GCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTT CAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAA TAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCAT TAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATG AAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATA GGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTG ATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTG ATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCA GATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTA AAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACAT ATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATT GAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGC GTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAG CATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAA AATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTAT GATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAG GTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAA GTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCAC TCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGA TAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGT GGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTAT TCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGA TTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGA GTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAG CAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTC TTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGA AACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAG TGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAG ACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGT AGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAA AATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAA AATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAAT CATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCT GGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATG TGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATA ACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTG AGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAG TTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAT ATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTG ATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTC TTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAG GAGGTGACCATCACCACCACCATCAC Cas9-NLS-6xHis (SEQ ID NO: 18): ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGC GGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATAC AGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGA GACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGA AGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATG ATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATG AACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATC CAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGC GCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGA GGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACA AACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTA AAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTC AGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGG GTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGC TTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATC AATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGA TATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAA ACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACA ACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAG GTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTG CTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGT GAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAAT CGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGG CGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCC CATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAAC GCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGT TTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTG AAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTC AAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAAT GCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTG GATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTT GAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGA TAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCG AAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTT GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTT GACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTAC ATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGA CTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAAT ATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTC GCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTC TTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATT ATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAA GTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAG ACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAA GTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAG TTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGT GAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACT AAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGA TAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTC CGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCAT GATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTT GAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCT AAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATC ATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCT CTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTT TGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAG AAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGAC AAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGT CCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTT TGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAG ACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAAC GGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGC AAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCA GAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGA GATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTT AGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAG CAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAA ATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGA TGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGT CAGCTAGGAGGTGACCCCAAGAAGAAGAGGAAGGTGATGGATAAGCATCACCACC ACCATCAC dCas9-VP64-6xHis (SEQ ID NO: 19): ATGGATAAGAAATACTCAATAGGCTTAGCTATCGGCACAAATAGCGTCGGATGGGC GGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATAC AGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGA GACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGA AGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATG ATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATG AACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATC CAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGC GCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGA GGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACA AACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTA AAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTC AGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGG GTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGC TTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATC AATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGA TATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAA ACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACA ACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAG GTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTG CTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGT GAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAAT CGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGG CGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCC CATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAAC GCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGT TTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTG AAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTC AAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAAT GCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTG GATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTT GAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGA TAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCG AAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTT GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTT GACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTAC ATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGA CTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAAT ATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTC GCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTC TTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATT ATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAA GTGATTATGATGTCGATGCCATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAG ACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAA GTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAG TTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGT GAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACT AAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGA TAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTC CGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCAT GATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTT GAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCT AAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATC ATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCT CTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTT TGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAG AAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGAC AAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGT CCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTT TGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAG ACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAAC GGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGC AAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCA GAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGA GATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTT AGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAG CAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAA ATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGA TGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGT CAGCTAGGAGGTGACGGTTCTCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAA AGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACA AGGCTGCAGGAGGCGGTGGAAGCGGGCGCGCCGACGCGCTGGACGATTTCGATCTC GACATGCTGGGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGAAGCGAC GCATTGGATGACTTTGATCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGAT CTCGATATGTTACATCACCACCACCATCAC

Example 2: Delivery of Cas9:gRNA in Adult Cochlea In Vivo

To study if Cas9 protein with gRNA can be delivered to adult cochlea in vivo, Cas9:gRNA-GFP complexed with lipofectamine2000 was microinjected in 1-month-old Atoh1-GFP mouse cochlea. Two weeks after injection disappearance of GFP signals in some outer hair cells (FIG. 1B, OHC in brackets) was observed whereas in control saline injected inner ear all OHCs have GFP signal (FIGS. 1A, 1B). OHCs without GFP could still be co-labeled with DAPI and Myo7a, a demonstration of survival of OHCs.

Cas9:gRNA Mediated Genome Editing in Pmca2 Mutant Mouse with Restoration of Hearing:

Mutation in Pmca2 gene (2765C→T) causes hereditary hearing loss in transgenic mouse (Oblivion, Obl) (Spiden et al., 2008; Street, McKee-Johnson, Fonseca, Tempel, & Noben-Trauth, 1998). To study if Cas9:gRNA can be used to disrupt the mutation to restore hearing, a set of gRNAs were designed, that target the mutation for Cas9:gRNA injection into neonatal Obl heterozygous (Pmca2^(Obl/+)) mouse inner ear at postnatal day 1 (P1) (FIG. 2; SEQ ID NOS: 21-32). A hearing study (ABR and DPOAE) was performed on the injected and uninjected inner ears, one, two and three months after injection.

In the uninjected cochlea, the hearing test showed profound hearing loss and the lack of DPOAE at the highest sound level used (90 dB) across all frequencies at one month of age, and hearing loss persisted throughout the rest of study (FIG. 3). In the injected mouse cochlea, drastic hearing improvement was observed, shown by ABR and DPOAE in all frequencies except for the highest frequency of 45.25 kHz. Hearing restoration was maintained in two and three month old cochleas injected with Cas9:gRNA (FIG. 3). Of all the gRNAs that targeted the mutation, only one (2.4-mut) showed hearing recovery, strongly supporting the importance in selecting gRNA with the specific effect (FIG. 4). To further evaluate the specificity of the study, gRNAs were designed against wildtype Pmca2 gene for inner ear injection (FIG. 2; SEQ ID NOS: 21-32). With wildtype gRNAs hearing improvement was not detected in the Pmca2^(Obl/+) cochlea injected animals. T7EI endonuclease assay was performed and showed that in vitro Cas9:gRNA cleaved mutant DNA (FIG. 5) at 125 nM. High-throughput sequencing (HTS) was performed to confirm that the strategy induces indels (insertions and deletions) in the injected inner ear, to provide molecular proof of genome editing.

To further correlate hearing recovery with hair cell status, injected and control inner ears were examined. By immunolabeling it was found that a majority of outer hair cells survived after injection whereas in control inner ear a majority of outer hair cells died especially in the base to middle turn (FIGS. 6A, 6B). In comparison to control, the injected inner ear maintained a large number neurites of spiral ganglion neurons (FIGS. 7A, 7B). Thus Cas9:gRNA mediated genome editing leads to outer hair cell survival and maintain their connection with neurons, resulting hearing recovery.

As mutations in PMCA2 gene are involved in human genetic deafness (Schultz, J. M. et al., 2005, N Engl J Med. 352(15), 1557-1564) this approach would lead to intervention in those patients. While this study is focused on the disruption of dominant mutation, it is highly feasible that a similar approach can be applied to recessive mutations when efficient for homology-directed repair (HDR) is improved.

An additional mechanism for hearing recovery in the injected inner ear could be due to interference of Cas9:gRNA that blocks of Pmca2 mutant transcription. Such interference has been previously reported (LRussa & Qi, 2015, Mol Cell Biol. doi.org/10.1128/MCB.00512-15). Interference by Cas9:gRNA to disrupt mutant gene that leads to functional recovery of hearing is a new mechanism that can be applied to other dominant hearing loss and genetic diseases in general.

Hearing Restoration in Tmc1 Beethoven (Bth) Genetic Hearing Loss Mouse Model:

To further study the generality of the Cas9:gRNA approach in restoring hearing, a Tmc1 mouse mutant (Bth) was used due to a dominant mutation (1235T→A that leads to M412K) (Vreugde, S., et al., (2002). Nat Genet, 30(3), 257-258), using the same procedure as outlined in Pmca2 study. A series of gRNA against the mutation as well as wildtype Tmc1 gene were designed. Tmc1 heterozygous mouse inner ear were injected with Cas9:gRNA at P1 and their hearing was assessed at 4 weeks of age. The uninjected inner ears served as control.

By ABR, a generally improved hearing was found across all frequencies with significant improvements at the frequencies of 11.32, 16 and 22.64 kHz. Over 20 dB improvements were seen in the latter two frequencies. For the Bth mice DPOAE were present. The injected ears had DPOAE similar to the uninjected inner ears with a slight elevation from 8 to 16 kHz (FIG. 8). In the Bth mice, hearing loss was largely due to dysfunctional inner hair cells, in contrast to Pmca2 mice where outer hair cells are affected. Combining Pmca2 and Tmc1 studies, it was demonstrated that this approach is suitable to both inner and outer hair cell related hearing loss. In human the same Tmc1 mutation (M412K) has been reported in a multi-generation family (Zhao Y. et al. PLoS ONE, 9(5), e97064. http://doi.org/10.1371/journal.pone.0097064). This study has potential to be developed as a treatment to be applied to those patients as a new therapy.

Example 3: Rescue of Hearing Loss by Cas9/gRNA Delivery In Vivo and CRISPR Mediated Gene Editing in a Genetic Deaf Mouse Model

To use liposomal formulation that complexes Cas9 with gRNA for CRISPR mediated gene editing as a potential treatment for genetic deafness, a rescue effect on Pmca2 deafness mouse mutant was studied. Pmca2 is a plasma membrane Ca²⁺ pump that is highly expressed in the inner ear hair cells, with the function that actively pumps out Ca²⁺ that enters hair cells during signal mechanoelectrical transduction during hearing and vestibular function. PMCA2 mutation has been shown to increase hearing loss severity human (M Schultz et al., “Modification of Human Hearing Loss by Plasma-Membrane Calcium Pump PMCA2,” N Engl J Med 352, no. 15 (Apr. 14, 2005): 1557-64, doi:10.1056/NEJMoa043899). In the mouse mutant (Oblivion) with a point mutation (S877F), severe to profound (i.e. complete) hearing loss is observed in heterozygous and homozygous mice (Spiden et al., PLoS Genetics 4, e1000238-e1000238.2008). This mouse mutant thus serves as an excellent model to determine if the Cas9/gRNA approach can be used to disrupt the Pmca2 mutation in heterozygous mice for hearing recovery, with implication to reduce hearing loss in human.

To study the hearing rescue effect, 12 guide RNAs were designed, 4 of which targeted the mutation. Following lipofectamine 2000 formulation that complexes Cas9 with each gRNA, the complex was injected into postnatal day 3 (P3) mouse cochleas. Both mouse mutants and wildtype control mice were injected. For each mouse right ear was injected and the left ear was uninjected. Three weeks or four weeks after injection, acoustic Auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) tests were performed.

For ABR and DPOAE tests, injected mice of either sex were anesthetized with xylazine (10 mg/kg, i.p.) and ketamine (100 mg/kg, i.p.). ABR and DPOAE were performed as previously described (Huang et al., 2013). ABR measures the auditory pathway from hair cells to brain; whereas DPOAE measures primarily outer hair cell function. By their combination it could be inferred if the hearing defects are of hair cells or central pathway deficiency.

At three weeks after injection, in the heterozygous Pmca2 mice, uninjected inner ears had profound hearing loss as shown by ABR and DPOAE. In the Cas9/gRNA-Pmca-2.4 (with the guide RNA 2.4) injected ears, significant hearing recovery in frequencies of 16, 22.64, 32 and 45.24 kHz by ABR was observed. By DPOAE, significant recovery in frequencies from 16 to 45.24 kHz was detected in the Cas9/gRNA-Pmca-2.4 injected inner ear, corresponding to ABR recovery. Recovery of DPOAE is an indication of restoration of hair cell function. To study long-term effect of hearing recovery, a hearing study was performed four weeks after injection and observed similar hearing recovery. The hearing study is continued at 6, 12 and 26 weeks after injection. In addition to the uninjected control ears, Pmca2 heterozygous mice injected with Cas9 complexed with other Pmca2 guide RNAs were also studied. No hearing recover was detected either by ABR or DPOAE was detected (data not shown). Thus guide RNA Pmca2-2.4 complexed with Cas9 induced sequence specific gene editing of Pmca2 mutation, leading to significant improvement of hearing.

To study potential toxicity associated with Cas9/gRNA delivery, Cas9/gRNA-Pmca-2.4 was injected into P3 wildtype (WT) mice and performed hearing study 3 weeks after injection. Slight elevation was observed in ABR and DPOAE at the highest frequency (45.24 kHz), but not in any other frequencies. Thus Cas9/gRNA-Pmca-2.4 complex does not cause additional damage to healthy hair cells or inner ear function. All together the study demonstrates that Cas9/gRNA that targets Pmca2 mutation in hair cells restores hearing in otherwise complete deaf mouse mutants. The similar strategy thus can be applied to human deaf patients with Pmca2 mutations to improve or restore hearing.

The hearing recovery in the Cas9/gRNA-Pmca-2.4 injected ear was not uniform across all frequencies (e.g. no recovery in 8 kHz). Further the recovery was uneven as better recovery was seen at the highest frequency of 45.24 kHz. This is likely due to the surgical procedure used only allowed for access to primarily the base of the cochlea, which is responsible for high-frequency hearing. The lack of recovery at the low frequency is likely due to the insufficient diffusion of Cas9/gRNA complex to the apical region of the cochlea. To test the hypothesis, additional experiments by multiple injections were performed in the Pmca2 mice over 6 days. By four weeks much greater hearing recovery was observed (40 dB) covering a majority of frequencies from 16 to 45.24 kHz (FIG. 9). The mouse inner ear is extremely small in size, about 1/50th of the size of human inner ear. While the mouse inner ear presents a surgical challenge in protein delivery, it is anticipated that in human inner ear the delivery would be considerably easier. Thus multiple injections result in greatly improved hearing restoration across most frequencies.

One of the most important applications of the technology is the ability to deliver the Cas9/gRNA complex in mature mammalian inner ear. The first set of experiments were conducted and showed that when injected into P9 mouse cochlea, a similar hearing rescue effect was observed (data not shown).

The work demonstrates the utility of direct Cas9/gRNA delivery into mammalian inner ear hair cells in vivo in disruption of mutations that leads to functional recovery of hearing. As 20% of genetic deafness is due to dominant mutations, this method can be tailored to target those mutations to restore hearing.

The most common form of deafness is recessive, for which repair of mutations will be needed for hearing restoration. One of the most common forms of deafness in human is age-related hearing loss (ARHL) or presbycusis, affecting over hundreds of millions of people worldwide. While the major mechanisms underlying ARHL is unknown, it is likely that genes will be identified with mutations or polymorphisms that make hair cells vulnerable to aging. Under this condition, the Cas9/gRNA could be applied to disrupt or repair the mutations/polymorphisms, to restore or slow down the progression of hearing loss. While the method currently targets hair cells, modifications will be made so that the method can be used to target inner ear cell types such as supporting cells, strial vascular and neurons, in which similar gene editing can be achieved for functional recovery of hearing. Finally many recessive genetic deafness is congenital, by the time of birth, simple gene editing may not be sufficient to restore cell function or hearing due to degeneration of the cell types. However it is possible to combine regeneration of the cell types with gene editing, to produce new cells while correcting mutations. These combinations can be applied to restore hearing in patients suffering from hearing loss due to different causes.

Example 4: Protein Delivery into the Inner Ear

The goal of this study was to use protein-mediated delivery system to deliver the biological proteins directly inner ear cell types with functional consequences. With this method, the proteins delivered have specific functions, and the effect is transient. Further, delivery of native protein lessens any potential immune response.

As cell surface is cationic (positively charged), it has been demonstrated that, by changing amino acids, GFP (green fluorescent protein) can be modified to be highly charged (both positively and negatively, named supercharged protein), which enables the supercharged GFP to enter cells. As a result, the super-charged GFP (s-GFP) can be used as vehicle to link and deliver other proteins into cells with biological effect in vitro and in vivo. This has been demonstrated in mouse retina in which s-GFP carrying Cre recombinase (s-GFP-Cre) was delivered that resulted in expression of reporter gene in retinal cells. The s-GFP used as (−30)-GFP, a negatively charged GFP protein.

The use of (−30)-GFP protein and a new positively charged (+36)-GFP protein to carry Cre, (−30)-GFP-Cre and (+36)-GFP-Cre, was tested for injection into mouse cochlea in vivo. The mouse used was Rosa-tdTomato^(f/f) in which functional Cre activity in the nuclei results in cells being labeled with tdT (red). It was shown that both (−30)-GFP-Cre and (+36)-GFP-Cre injections led to tdT expression in the cochlear hair cells. Near the injection site (base turn of cochlea), ˜30% of hair cells became tdT-labeled (FIGS. 10A, 10B), The study demonstrated that supercharged GFP proteins can be used to delivery protein directly into inner ear hair cells with biological effect in nuclei.

It was next tested whether Aurein, an antimicrobial peptide, in combination with s-GFP, can serve as an effective carrier to deliver functional proteins into mouse inner ear cell types with high efficiency. (+36)-GFP-aurein-Cre was injected with different concentrations into P3 Rosa-tdT mouse inner ear by cochleostomy in vivo. The tissues were harvested 5 days later for immunolabeling to identify tdT⁺ inner ear cell types. Overall it was found that cochlear hair cells were primarily labeled with tdT, with additional tdT labeling in supporting cells and in spiral ganglion neurons. At low concentrations of 5 μM, few IHC and some supporting cells (SC) were tdT⁺. At 22.5 μM, 58% of OHC and 75% of IHC, as well as 8% of SC were tdT⁺. At the highest 50 μM, 96% of OHC, 88% of IHC and 19% of SC were tdT⁺. Some spiral ganglion neurons were also tdT at 22.5 and 50 μM (FIGS. 11A-11E and FIG. 12). There was slight cell toxicity associated with the highest concentration of 50 μM. It was found that 20% of IHC loss 8% of OHC in the injected animals, whereas at lower concentrations of 5 and 22.5 μM no cell loss was detected.

The study demonstrated that functional proteins including nuclear proteins can be conjugated with supercharged protein (+36)GFP fused with aurein. The protein complex was directly injected into mammalian inner ear that leads to uptake by a wide range of inner ear cell types. The protein delivered was properly localized in the nuclei and had the specific biological function in cleaving floxP sites on the DNA sequence, to activate tdTomato in the inner ear cells with high efficiency.

The delivery system can be used to study protein functions, inner ear regeneration for hearing recovery, hearing protection, and gene editing to correct genetic deafness by genome-editing proteins.

Example 5: Characterization of a Peptide that Enhances Endosomal Escape of Delivered Proteins In Vitro and In Vivo

In this study, aurein 1.2 (GLFDIIKKIAESF; SEQ ID NO: 34) was discovered as a peptide that enhances the endosomal escape of a variety of cargo fused to +36 GFP. The structure-function relationships were elucidated within aurein 1.2 using alanine scanning and mutational analysis. Results from three independent delivery assays confirmed that treatment of mammalian cells with cargo proteins fused to aurein 1.2-+36 GFP result in more efficient cytosolic delivery than the same proteins fused to +36 GFP alone. The ability of aurein 1.2 to enhance non-endosomal protein delivery in vivo is also described. Cre recombinase enzyme was delivered into hair cells in the cochlea (inner ear) of live mice with much greater (>20-fold) potency when fused with aurein 1.2 than in the absence of the peptide. These results together provide a simple molecular strategy for enhancing the cytosolic delivery of proteins in cell culture and in vivo that is genetically encoded, localized to cargo molecules, and does not require global treatment with cytotoxic small molecules.

Materials and Methods

Expression and Purification of Proteins.

E. coli BL21 STAR (DE3) competent cells (Life Technologies) were transformed with pET29a expression plasmids. Colonies from the resulting expression strain was directly inoculated in 1 L of Luria-Bertani (LB) broth containing 100 μg/mL of ampicillin at 37° C. to OD₆₀₀=˜1.0. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce expression and the culture was moved to 20° C. After ˜16 h, the cells were collected by centrifugation at 6,000 g and resuspended in lysis buffer (Phosphate buffered saline (PBS) with 1 M NaCl). The cells were lysed by sonication (1 sec pulse-on, 1 sec pulse-off for 6 min, twice, at 6 W output) and the soluble lysate was obtained by centrifugation at 10,000 g for 30 min.

The cell lysate was incubated with His-Pur nickel-nitriloacetic acid (Ni-NTA) resin (Thermo Scientific) at 4° C. for 45 min to capture His-tagged protein. The resin was transferred to a 20-mL column and washed with 20 column volumes of lysis buffer plus 50 mM imidazole. Protein was eluted in lysis buffer with 500 mM imidazole, and concentrated by Amicon ultra centrifugal filter (Millipore, 30-kDa molecular weight cut-off) to ˜50 mg/mL. The eluent was injected into a 1 mL HITRAP SP HP column (GE Healthcare) after dilution into PBS (5-fold). Protein was eluted with PBS containing a linear NaCl gradient from 0.1 M to 1 M over five column volumes. The eluted fractions containing protein were concentrated to 50 μM as quantified by absorbance at 488 nm assuming an extinction coefficient of 8.33×10⁴ M⁻¹ cm⁻¹ as previously determined (McNaughton, B. R.; et al. Proceedings of the National Academy of Sciences 2009, 106, 6111), snap-frozen in liquid nitrogen, and stored in aliquots at −80° C.

Cell Culture.

All cells were cultured in Dulbecco's modification of Eagle's medium (DMEM w/glutamine, Gibco) with 10% fetal bovine serum (FBS, Gibco), 5 I.U. penicillin, and 5 g/mL streptamycin. All cells were cultured at 37° C. with 5% CO₂.

Peptide Synthesis.

Peptides were ordered from ChinaPeptides Co., LTD, each 4 mg, purity >90%. HPLC and MALDI data were provided with lyophilized peptides. Peptides were resuspended in DMSO to a final concentration of 10 mM.

Sortase Conjugation.

All reactions were performed in 100 mM Tris buffer (pH 7.5) with 5 mM CaCl₂ and 1M NaCl. For peptide conjugation to the N-terminus of GGG-+36-GFP, 20 μM of protein with N-terminal Gly-Gly-Gly was incubated with 400 μM of peptide with C-terminal LPETGG and 1 μM eSrtA for 2 h at room temperature in a 50 μL reaction. The unreacted peptides were removed through spin filtration with an Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore, 30-kDa molecular weight cut-off). The reaction mixture was washed twice with 500 μL of buffer each time to a final concentration of 50 μL. Conjugation efficiency was determined through LC-MS (Agilent 6220 ESI-TOF) using protein deconvolution through MaxEnt (Waters) by comparing relative peak intensities.

For conjugation of GGGK^(Dex) to +36-GFP-LPETG-His₆, 10 μM of protein was incubated with 400 μM of peptide and 2 μM eSrtA at room temperature. The reaction was quenched with 10 mM ethylenediaminetetraacetic acid (EDTA) after 2 h. For aurein 1.2-+36-GFP-LPETG-His₆, a N-terminal His₆-ENLYFQ was added to prevent sortase reaction with the N-terminal glycine of aurein 1.2. The N-terminal tag was removed with 200 μM TEV protease at 4° C. for 16 h to release the native N-terminal sequence of aurein 1.2-+36-GFP. Successful conjugation of GGGK^(Dex) removes the C-terminal His₆ tag and allows for purification through reverse Ni-NTA column. Unreacted protein binds to the Ni-NTA, and the unbound protein was collected and concentrated as described above.

Plasmid Transfection.

Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies) according the manufacturer's protocol.

Synthesis of Dexamethasone-21 Thiopropionic Acid (SDex).

Synthesis of dexamethasone-21-mesylate was performed as previously described (Simons, S. S.; et al. J Org Chem 1980, 45, 3084; Dunkerton, L. V.; et al. Steroids 1982, 39, 1). 2 g of dexamethasone stirring in 38 mL anhydrous pyridine under nitrogen was reacted with 467.2 mg methanesulfonyl chloride (1.2 eq.) on ice for 1 h, after which another 311 methanesulfonmethanesulfonyl chloride was added and allowed to react overnight (16 h) on ice. Next, the reaction was added to 800 mL of ice water and Dexamethasone-21-Mesylate (Dex-21-OMs) formed a white precipitate. The slurry was filtered and the precipitate washed with 800 mL of ice water, dried under high vacuum overnight and quantified by LC-MS (m/z 471.19 Da, 83% yield).

Dexamethasone-21-thiopopionic acid (S^(Dex)) was prepared as previously described (Kwon, Y. U.; Kodadek, T. J Am Chem Soc 2007, 129, 1508). 2.05 g of Dex-21-OMs was added to 2 eq. thiopropionic acid and 4 eq. triethylamine stirring in anhydrous acetone at room temperature overnight. The following morning, the reaction was added to 800 mL of ice water and acidified with 1N HCl until S^(Dex), visible as an off-white solid, precipitation was complete. The mixture was filtered, washed with 800 mL ice cold water acidified to pH 1 with HCl, dried under high vacuum overnight and analyzed by LC-MS (m/z 481.21 Da, 63% yield).

Synthesis and Purification of GGGK^(Dex).

GGGK^(Dex) was synthesized on Fmoc-Lys (Mtt)-Wang resin (BACHEM, D-2565) using microwave acceleratin (MARS, CEM). Coupling reactions were performed using 5 equivalents of Fmoc-Gly-OH (Novabiochem, 29022-11-5), 5 equivalents of PyClock (Novabiochem, 893413-42-8) and 10 equivalents of diisopropylethylamine (DIEA) in N-methylpyrrolidone (NMP). Fmoc groups were removed using 25% piperidine in NMP (efficiency quantified; ε₂₉₉=6234 M⁻¹ cm⁻¹ in acetonitrile) and Mtt groups were removed by incubating the Fmoc-GGGK(Mtt)-resin with 2% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 20 min, after which the resin was washed with 2% TFA in DCM until the characteristic yellow color emitting from the Mttcation subsided. After Mtt removal, S^(Dex)—COOH (Dex-21-thiopropinonic acid) was coupled to the Nε of the lysine side-chain by incubating the Fmoc-GGGK-resin with 2.5 eq. S^(Dex)—COOH, 2.5 eq. HATU, 2.5 eq. HOAt, 5 eq. DIEA and 5 eq. 2,6-lutidine in 2.5 mL NMP overnight, at room temperature, on an orbital shaker. After S^(Dex)-labeling, Fmoc-GGGK^(Dex)-resin was washed thoroughly with NMP and DCM, the N-terminal Fmoc was removed using 25% piperidine in NMP, and crude peptides were dissociated from the resin by incubating the GGGK^(Dex)-resin in a cleavage cocktail composed of 81.5% trifluoroacetic acid (TFA), 5% thioanisole, 5% phenol, 5% water, 2.5% ethanedithiol and 1% triisopropylsilane for 30 min at 38° C. Crude peptides were precipitated in 40 mL cold diethyl ether, resuspended in water, lyophilized and purified via reverse phase high-pressure liquid chromatography (HPLC) using a linear gradient of acetonitrile and water with 0.1% TFA across a C18 (VYDAC, 250 mm×10 mm ID) column. Purified peptides were lyophilized and stored at 4° C. Polypeptide identity was confirmed by mass spectrometry on a Waters QToF LC-MS, and purity was measured by analytical reverse-phase HPLC (Shimadzu Instruments) using a C18 column (Poroshell 120 SB—C18, 2.7 μm, 100 mm×3 mm ID, Agilent).

Image Processing for Primary Screen.

BSR.LNL.tdTomato cells were plated at 10,000 cells per well in black 384-well plates (Aurora Biotechnologies). Cells were treated with Cre fusion proteins diluted in serum-free DMEM 24 h after plating and incubated for 4 h at 37° C. Following incubation, the cells were washed three times with PBS+20 U/mL heparin. The cells were incubated a further 48 h in serum-containing media. Cells were fixed in 3% paraformaldehyde and stained with Hoescht 33342 nuclear dye. Images were acquired on an ImageXpress Micro automated microscope (Molecular Devices) using a 4× objective (binning 2, gain 2), with laser- and image-based focusing (offset −130 μm, range ±50 μm, step 25 μm). Images were exposed for 10 ms in the DAPI channel (Hoechst) and 500 ms in the dsRed channel (tdTomato). Image analysis was performed using the cell-scoring module of MetaXpress software (Molecular Devices). All nuclei were detected with a minimum width of 1 pixel, maximum width of 3 pixels, and an intensity of 200 gray levels above background. Positive cells were evaluated for uniform signal in the dsRed channel (minimum width of 5 pixels, maximum width of 30 pixels, intensity >200 gray levels above background, 10 μm minimum stained area). In total, nine images were captured and analyzed per well, and 16 wells were treated with the same fusion protein. The primary screen was completed in biological triplicate.

Cre Delivery Assay.

Uptake and delivery assays for Cre fusion proteins were performed as previously described (Cronican, J. J.; et al. ACS Chemical Biology 2010, 5, 747). Briefly, proteins were diluted in serum-free DMEM and incubated on the cells in 48-well plates for 4 h at 37° C. Following incubation, the cells were washed three times with PBS+20 U/mL heparin. The cells were incubated a further 48 h in serum-containing media prior to trypsinization and analysis by flow cytometry. All flow cytometry were carried out on a BD Fortessa flow cytometer (Becton-Dickinson) using 530/30 nm and 610/20 nm filter sets. Toxicity for aurein 1.2 and citropin 1.3 validation assays was determined using CELLTITER-GLO assay (Promega) in 96-well plates following manufacturer protocol. Toxicity for alanine scan mutational analysis was determined with LIVE/DEAD fixable far-red dead cell stain (Life Technologies) with 635 nm laser and 670/30 nm filter.

GR-mCherry Translocation Assay.

One day prior to transfection 10,000 HeLa cells in 200 μL of DMEM (10% FBS, 1×Pen-Strep) were plated into single wells of a 96-well MATRICAL glass bottom microplate (MGB096-1-2-LG-L) and allowed to adhere overnight. The following day, cells were transfected with GRmCherry using LIPOFECTAMINE® 2000 technologies. Following transfection, cells were allowed to recover overnight in DMEM (+10% FBS). The following day, cells were treated with dexamethasone (Dex) or 1 μM Dex-protein conjugate in the presence or absence of inhibitor diluted into DMEM (without phenol red, +300 nM Hoescht33342). After 30 min, cells were washed twice with 200 μL of HEPES-Krebs-Ringer's (HKR) buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES at pH 7.4), after which 100 μL of HKR buffer was overlaid onto the cells and images were acquired on a Zeiss Axiovert 200M epifluorescence microscope outfitted with Ziess AxiocammRM camera and an EXFO-Excite series 120 Hg arc lamp. The translocation ratio (the ratio of median GFP intensity in the nuclear and surrounding regions) for individual cells was measured using CELLPROFILER® as described 36. To examine the effect of endocytosis inhibitors, HeLa cells were pretreated for 30 min with DMEM (without phenol red) containing inhibitors (80 μM Dynasore, 5 mM MBCD, 50 μM EIPA, 200 nM bafilomycin or 200 nM wortmannin) at 37° C. for 30 min before incubation with Dex or Dex-protein conjugates.

BirA Translocation Assay.

One day prior to transfection, 100,000 HeLa cells in 1 mL of DMEM (10% FBS, 1×PenStrep) were plated into single wells of a 12-well tissue culture plate and allowed to adhere overnight. Cells were transfected with mCherry-AP fusion protein using LIPOFECTAMINE® 2000 technologies according to manufacture guidelines 24 h before protein treatment. Next day, transfected cells were treated for 1 h at 37° C. with +36 GFP-BirA or aurein 1.2-+36 GFP-BirA diluted in serum-free DMEM at 250 nM, 500 nM and 1 μM concentrations. 250 nM+36 GFP-BirA+100 μM chloroquine was also used as a positive control for endosomal escape. The cells were washed three times with PBS containing heparin to remove excess supercharged proteins that were not internalized. The cells were then treated with 100 μL of 10 μM biotin and 1 mM ATP in PBS for 10 min. The reaction was quenched with excess (10 μL of 8 mM) synthesized AP before cells were trypsinized and lysed. To verify that extracellular BirA was not generating signal during lysis, 1 μM+36 GFP-BirA or aurein 1.2-+36 GFP-BirA was added during the quench step to untreated wells. Cells were lysed with 100 μL of trypsin and lysed with QIASHREDDER columns (Qiagen). 30 μL of lysate was loaded onto 4-12% Bis-Tris Bolt gels in Bolt-MES buffer (Life Technologies) and ran for 20 min at 200 volts. Gels were transferred to PVDF membrane using IBLOT2 transfer system (Life Technologies). Biotinylation was measured through western blotting using the LI-COR quantitative infrared fluorescent antibodies and the Odyssey Imager detection system. To normalize for transfection and gel loading variables, the ratio of biotin signal to mCherry signal was used for comparison.

Cytosolic Fractionation Assay.

One day prior to fractionation, 4×10⁶ HeLa cells were plated in 20 mL of DMEM (10% FBS, 1×PenStrep, no phenol red) in 175-cm² culture flasks and allowed to adhere for 15 hours. The following day, the media was removed from each flask and the cells were washed twice with clear DMEM (no FBS, no PenStrep, no phenol red). The media was replaced with 7 mL of clear DMEM containing +36 GFP or aurein 1.2-+36GFP at a concentration of 250 nM, 500 nM, or 1 μM. Several flasks were treated with clear DMEM to be used as negative controls and to generate calibration curves with the cytosolic extracts. The cells were incubated for 30 min at 37° C., 5% CO₂ after which they were washed three times with PBS. Using a cell-scraper, the cells were suspended in 5 mL of PBS, transferred into a 15 mL Falcon tube, and pelleted at 500 g for 3 min. The cells were resuspended in 1 mL PBS, counted using an automated cell counter (Auto T4, CELLOMETER®), and pelleted again at 500 g for 3 min. The cell pellet was resuspended in ice-cold isotonic sucrose (290 mM sucrose, 10 mM imidazole, pH 7.0 with 1 mM DTT, and COMPLETE™, EDTA-free protease inhibitor cocktail) and transferred to a glass test tube on ice. The cells were homogenized with an Omni TH homogenizer outfitted with a stainless steel 5 mm probe for three 30 s pulses on ice with 30 s pauses between the pulses. The homogenized cell lysate was sedimented at 350 Kg in an ultracentrifuge (TL-100; Beckman Coulter) for 30 min at 4° C. using a TLA 120.2 rotor. The supernatant (cytosolic fraction) was analyzed in a 96-well plate on a fluorescence plate reader (SYNERGY 2, BioTek, excitation=485+/−10 nm, emission=528+/−10 nm). The concentration of the protein conjugate in the cytosol was determined using a standard curve relating fluorescence to known protein concentrations. To generate the standard curve, known concentrations of +36 GFP and aurein 1.2-+36 GFP between 0.2 nM and 1 μM were added to cytosolic extracts of the untreated negative controls. For background subtraction, several wells containing cytosolic extracts from untreated cells were averaged, and this average was subtracted from each well.

Total Protein Delivery Assay.

One day prior to the experiment, 100,000 HeLa cells/well were plated in DMEM (10% FBS, lx PenStrep, no phenol red) in 6-well plates and allowed to adhere for 15 hours. The following day, the media was removed from each well and the cells were washed twice with clear DMEM (no FBS, no PenStrep, no phenol red). The media was replaced with 1 mL of clear DMEM containing +36 GFP or aurein 1.2-+36 GFP at concentrations of 250 nM, 500 nM, or 1 μM. The cells were incubated for 30 min at 37° C., 5% CO₂ after which they were washed three times with PBS containing 20 U/mL heparin (Sigma) to remove surface-bound cationic protein. The cells were trypsinized for 5 min, pelleted in serum-containing DMEM for 3 min at 500 g, washed with 1 mL PBS, and pelleted again for 3 min at 500 g. The cell pellet was resuspended in 100 μL PBS. Flow cytometry was performed on a BD Accuri C6 Flow Cytometer at 25° C. Cells were analyzed in PBS (excitation laser=488 nm, emission filter=533+/−30 nm). At least 10,000 cells were analyzed for each sample. For background subtraction, wells were treated with clear DMEM only. The average of three untreated wells was subtracted from each +36 GFP conjugate-containing well.

Microinjection of Proteins to Mouse Inner Ear.

P1-2 Gt(ROSA)26Sor^(tm14(CAG-tdTomato)Hze) mice were used for aurein 1.2-+36-GFP-Cre and +36-GFP-Cre injection. The Rosa26-tdTomato mice were from the Jackson Laboratory. Animals were used under protocols approved by the Massachusetts Eye & Ear Infirmary IACUC committee. Mice were anesthetized by hypothermia on ice. Cochleostomies were performed by making an incision behind the ear to expose the cochlea. Glass micropipettes held by a micromanipulator were used to deliver the complex into the scala media, which allows access to inner ear hair cells. The total delivery volume for every injection was 0.4 μL per cochlea and the release was controlled by a micromanipulator at the speed of 69 nL/min.

Immunohistochemistry and Quantification.

5 days after injection, the mice were sacrificed and cochlea were harvested by standard protocols (Sage, C.; et al. Science 2005, 307, 1114). For immunohistochemistry, antibodies against hair-cell markers (Myo7a) and supporting cells (Sox2) were used following a previously described protocol (Sage C et al., supra). To quantify the number of tdTomato positive cells after aurein 1.2-+36-GFP-Cre and +36-GFP-Cre, we counted the total number of inner and outer hair cells in a region spanning 100 μm in the apex, middle, and base turn of the cochlea.

Results

Preparation of Antimicrobial Peptide (AMP) Conjugates of Supercharged GFP-Cre Fusion Protein:

AMPs from the Antimicrobial Peptide Database (Wang, Z.; Wang, G. Nucleic acids research 2004, 32, D590) that are ≦25 amino acids long (to facilitate their preparation and conjugation to +36 GFP), lack post-translational modifications, and are not known to be toxic to mammalian cells. Based on these criteria, 36 AMPs were identified ranging from 9 to 25 amino acids in length (Table 1). Each of the peptides was synthesized on solid phase with an LPETGG (SEQ ID NO: 89) sequence appended to their C-terminus to enable sortase-catalyzed conjugation (Chen, I.; et al. Proceedings of the National Academy of Sciences 2011, 108, 11399) (FIG. 13B). Assembly of proteins using sortase proved more amenable to rapid screening than the construction and expression of the corresponding fusions, especially since several AMP fusions do not express efficiently in E. coli.

The peptides were conjugated to purified GGG-(+36 GFP)-Cre using the previously described evolved sortase A enzyme (eSrtA) (Chen, I.; et al. Proceedings of the National Academy of Sciences 2011, 108, 11399). Sortase catalyzes the transpeptidation between a substrate containing the C-terminal LPETGG (SEQ ID NO: 89) and a substrate containing an N-terminal glycine to form a native peptide bond linkage and a protein identical to the product of translational fusion.

TABLE 1 List of peptides chosen from the Antimicrobial Peptide Database (APD) Conju- gation APD effici- Label number Sequence ency A AP00408 FLFPLITSFLSKVL 55% (SEQ ID NO: 36) B AP00405-11 FISAIASMLGKFL 70% (SEQ ID NO: 37) C AP00327 GWFDVVKHIASAV — (SEQ ID NO: 38) D AP01434 FFGSVLKLIPKIL — (SEQ ID NO: 39) E AP00013 GLFDIIKKIAESF 77% (SEQ ID NO: 34) F AP00025 HGVSGHGQHGVHG 20% (SEQ ID NO: 40) G AP00094 FLPLIGRVLSGIL — (SEQ ID NO: 41) H AP00012 GLFDIIKKIAESI 28% (SEQ ID NO: 42) I AP00014 GLLDIVKKVVGAFGSL — (SEQ ID NO: 43) J AP00015 GLFDIVKKVVGALGSL 13% (SEQ ID NO: 44) K AP00016 GLFDIVKKVVGAIGSL — (SEQ ID NO: 45) L AP00017 GLFDIVKKVVGTLAGL 18% (SEQ ID NO: 46) M AP00018 GLFDIVKKVVGAFGSL — (SEQ ID NO: 47) N AP00019 GLFDIAKKVIGVIGSL — (SEQ ID NO: 48) O AP00020 GLFDIVKKIAGHIAGSI — (SEQ ID NO: 49) P AP00021 GLFDIVKKIAGHIASSI — (SEQ ID NO: 50) Q AP00022 GLFDIVKKIAGHIVSSI — (SEQ ID NO: 51) R AP00101 FVQWFSKFLGRIL 51% (SEQ ID NO: 52) S AP00351 GLFDVIKKVASVIGGL 11% (SEQ ID NO: 53) T AP00352 GLFDIIKKVASVVGGL — (SEQ ID NO: 54) U AP00353 GLFDIIKKVASVIGGL 4% (SEQ ID NO: 35) V AP00567 VWPLGLVICKALKIC 4% (SEQ ID NO: 55) W AP00597 NFLGTLVNLAKKIL 34% (SEQ ID NO: 56) X AP00818 FLPLIGKILGTIL 14% (SEQ ID NO: 57) Y AP00866 FLPIIAKVLSGLL 86% (SEQ ID NO: 58) Z AP00870 FLPIVGKLLSGLL — (SEQ ID NO: 59) AA AP00875 FLSSIGKILGNLL 88% (SEQ ID NO: 60) AB AP00898 FLSGIVGMLGKLF 70% (SEQ ID NO: 61) AC AP01211 TPFKLSLHL 81% (SEQ ID NO: 62) AD AP01249 GILDAIKAIAKAAG 20% (SEQ ID NO: 63) AE AP00013- LFDIIKKIAESF 63% G (SEQ ID NO: 64) AF AP00013- LFDIIKKIAESGFLFDIIKKIAESF — 2x (SEQ ID NO: 65) AG AP00722- GLLNGLALRLGKRALKKIIKRLCR — 75 (SEQ ID NO: 66) AH His13 GHHHHHHHHHHHHH — (SEQ ID NO: 67) AI AP00512 FKCRRWQWRM 42% (SEQ ID NO: 68) AJ AP00553 KTCENLADTY — (SEQ ID NO: 69) Peptides were synthesized with a C-terminal LPETGG tag to enable conjugation with an evolved sortase (eSrtA). Conjugation efficiencies were calculated based on LC-MS results using peak abundance as determined through MaxEnt protein deconvolution.

The efficiency of eSrtA-mediated conjugation varied widely among the peptides. Of the 36 peptides chosen for screening, 20 showed detectable (4% to 88%) sortase-mediated conjugation to +36 GFP-Cre, as observed by LC-MS, to generate desired peptide-LPETGGG-(+36 GFP)-Cre fusion proteins (Table 1). Unreacted peptide was removed by ultrafiltration with a 30-kD molecular weight cut off membrane.

Primary Screen for Endosomal Escape.

The ability of each peptide-(+36 GFP)-Cre recombinase fusion was assayed when added to culture media to effect recombination in BSR.LNL.tdTomato cells (Cronican, J. J.; et al. ACS Chemical Biology 2010, 5, 747), a hamster kidney cell line derived from BHK-21. Because Cre recombinase must enter the cell, escape endosomes, enter the nucleus, and catalyze recombination to generate tdTomato fluorescence, this assay reflects the availability of active, non-endosomal recombinase enzyme that reaches the nucleus. As a positive control, cells were treated with +36 GFP-Cre and chloroquine, a known endosome-disrupting small molecule (Dijkstra, J.; et al. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1984, 804, 58).

The reporter BSR.LNL.tdTomato cells were incubated with 250 nM of each peptide-(+36 GFP)-Cre protein in serum-free media. In the absence of any conjugated peptide, treatment of reporter cells with 250 nM +36 GFP-Cre protein resulted in 4.5% of the cells expressing tdTomato, consistent with previous reports 18. The same concentration of protein incubated with 100 μM chloroquine as a positive control resulted in an average of 48% recombined cells (FIG. 14). The results of chloroquine treatment varied substantially between independent replicates. As chloroquine is known to be toxic to cells above 100 μM, it was speculated that this variability arises from the small differences between chloroquine's efficacious and toxic dosages.

Ten of the screened peptide conjugates resulted in recombination efficiencies that were significantly above that of +36 GFP-Cre (FIG. 14). The most potent functional delivery of Cre was observed with aurein 1.2-+36 GFP-Cre (Table 1, entry E). Treatment with aurein 1.2-+36 GFP-Cre resulted in an average of 40% recombined cells, comparable to that of the chloroquine positive control (FIG. 14). To investigate the impact of differential conjugation efficiency on peptide performance, citropin 1.3 (Table 1, entry U), which displayed a moderate level of recombination and the lowest level of conjugation (4%), was compared to aurein 1.2, which has the highest level of recombination and also a high level of conjugation (77%).

Both aurein 1.2-+36 GFP-Cre and citropin 1.3-+36 GFP-Cre were cloned, expressed, and purified as fusion proteins. The recombination signal from treatment with 250 nM of expressed aurein 1.2-+36 GFP-Cre was 10.4-fold above that of +36 GFP-Cre. In contrast, treatment with 250 nM expressed citropin 1.3-+36 GFP-Cre did not induce any enhanced Cre delivery. When the treatment concentration was increased to 1 μM, aurein 1.2-+36 GFP-Cre and citropin 1.3-+36 GFP-Cre resulted in 3.8-fold and 3.0-fold higher recombination levels, respectively, than that of +36 GFP-Cre alone (FIG. 15A). These results evidence that while aurein 1.2 and citropin 1.3 both enhance the delivery of functional, non-endosomal +36 GFP-Cre protein at high concentrations, aurein 1.2 has greater efficacy than citropin 1.3 at lower concentrations.

Next, the toxicity of each fusion protein was evaluated at a range of concentrations (125 nM to 1 μM) using an ATP-dependent cell viability assay at 48 h after treatment. For +36 GFP-Cre, it was observed that there was no cellular toxicity up to 1 μM treatment, which resulted in 85% viable cells. Cells treated with 250 nM recombinant aurein 1.2-+36 GFP-Cre and citropin 1.3-+36 GFP-Cre displayed 87% and 84% viability, respectively. Applying 1 μM treatments decreased cell viability to 70% and 66%, respectively (FIG. 15B). In light of its activity and low cytotoxicity at 250 nM, the ability of aurein 1.2 to enhance cytosolic protein delivery was characterized in depth.

Site-Directed Mutagenesis of Aurein 1.2.

Aurein 1.2 (GLFDIIKKIAESF; SEQ ID NO: 34) is a potent AMP excreted from the Australian tree frog, Litoria aurea (Rozek, T.; et al. Rapid Communications in Mass Spectrometry 2000, 14, 2002). Interestingly, citropin 1.3 (GLFDIIKKVASVIGGL; SEQ ID NO: 35) is a closely related peptide and is excreted from a different Australian tree frog, Litoria citropa (Wegener, K. L.; et al. European journal of biochemistry/FEBS 1999, 265, 627). While the properties of aurein 1.2 have been investigated for its anti-bacterial and anti-tumorigenic abilities ((Rozek, T.; et al. Rapid Communications in Mass Spectrometry 2000, 14, 2002), its ability to enhance endosomal escape or macromolecule delivery has not been previously reported. The free peptide is thought to adopt an amphipathic alpha helical structure in solution, but the length of the helix is too short to span a lipid bilayer (Balla, M.; Bowie, J. H.; Separovic, F. European Biophysics Journal 2004, 33, 109. Therefore it was theorized that aurein 1.2 disrupts membranes through a “carpet mechanism” in which peptides bind to the membrane surface in a manner that allows hydrophobic residues to interact with lipid tails and hydrophilic residues to interact with polar lipid head groups (Fernandez, D. I.; et al. Physical Chemistry Chemical Physics 2012, 14, 15739). Above a critical concentration, the peptides are thought to alter the curvature of the membrane enough to break apart the compartment.

TABLE 2 Site-directed mutagenesis of aurein 1.2 Label Sequence Aurein 1.2 GLFDIIKKIAESF (SEQ ID NO: 34) G1A ALFDIIKKIAESF (SEQ ID NO: 70) L2A GAFDIIKKIAESF (SEQ ID NO: 71) F3A GLADIIKKIAESF (SEQ ID NO: 72) D4A GLFAIIKKIAESF (SEQ ID NO: 73) I5A GLFDAIKKIAESF (SEQ ID NO: 74) I6A GLFDIAKKIAESF (SEQ ID NO: 75) K7A GLFDIIAKIAESF (SEQ ID NO: 76) K8A GLFDIIKAIAESF (SEQ ID NO: 77) I9A GLFDIIKKAAESF (SEQ ID NO: 78) E11A GLFDIIKKIAASF (SEQ ID NO: 79) S12A GLFDIIKKIAEAF (SEQ ID NO: 80) F13A GLFDIIKKIAESA (SEQ ID NO: 81) K7H GLFDIIHKIAESF (SEQ ID NO: 82) K8H GLFDIIKHIAESF (SEQ ID NO: 83) E11H GLFDIIKKIAHSF (SEQ ID NO: 84) K7R GLFDIIRKIAESF (SEQ ID NO: 85) K8R GLFDIIKRIAESF (SEQ ID NO: 86) E11R GLFDIIKKIARSF (SEQ ID NO: 87) E11D GLFDIIKKIADSF (SEQ ID NO: 88)

-   -   An alanine scan was performed on aurein 1.2 to determine         positions that tolerate mutation. Charged amino acids at         tolerant positions were then replaced with histidines or other         charged amino acids in an attempt to increase endosomal escape         efficiency. All constructs were expressed as recombinant fusion         proteins with +36 GFP-Cre.

To identify the residues involved in enhancing non-endosomal protein delivery, an alanine scan of the 13 amino acid positions of aurein 1.2 was performed, by cloning, expressing, and purifying each alanine mutant of aurein 1.2-+36 GFP-Cre. The resulting fusion proteins were assayed in BSR.LNL.tdTomato reporter cells as described above (Table 2). Seven positions were moderately to highly intolerant of alanine substitution. Six positions retained >70% of the activity of unmutated aurein 1.2-+36 GFP-Cre (FIG. 16A). At each of these tolerant positions, which included three positions with charged residues (K7, K8, and E11 from Table 2), additional mutations were generated in an effort to improve activity. In total, 19 mutants of aurein 1.2 were generated and tested using the Cre recombination assay. Two of the aurein variants, K8R and S12A, exhibited potentially improved overall recombination efficiency but also increased toxicity at 250 nM (FIG. 16B). Given this increase in toxicity, it was decided to focus on the original peptide, aurein 1.2, and proceeded to characterize its potency through a series of complementary secondary assays.

Independent Assays of Endosomal Escape.

Although endosomal escape is widely considered to be the major bottleneck of cationic protein delivery (Sahay, G.; et al. Nature Biotechnology 2013, 31, 653), few assays quantify the ability of proteins to escape endosomes on a single-cell basis. To quantify cytosolic delivery of supercharged proteins in individual cells, a glucocorticoid receptor (GR) translocationassay (Yu, P.; Liu, B.; Kodadek, T. Nat Biotech 2005, 23, 746) was applied. In untreated HeLa cells expressing mCherry-labeled GR (GR-mCherry), the GR distributes nearly uniformly throughout the cell interior, resulting in a nuclear-to-cytoplasm translocation ratio (TR) of 1.17 (FIGS. 17A and 17B). Upon treatment with the cell-permeable glucocorticoid dexamethasone-21-thiopropionic acid (S^(Dex)) at a concentration of 1 μM for 30 min, GR-mCherry relocates almost exclusively to the nucleus, resulting in a TR of 3.77 (FIGS. 17A and 17B).

Dexamethasone conjugates of +36 GFP (+36 GFP^(Dex)) and aurein 1.2-+36 GFP (aurein 1.2-+36 GFP^(Dex)) were generated via sortase-mediated conjugation. Conjugated to these proteins, S^(Dex) is no longer cell permeable and cannot activate the GR for nuclear translocation unless the protein-S^(Dex) conjugate can access the cytosol. Treatment of HeLa cells expressing GR-mCherry with 1 μM aurein 1.2-+36 GFP^(Dex) for 30 min resulted in a TR of 2.62, which was significantly greater (p<0.05) than that of +36 GFP^(Dex) (TR=2.23). As positive controls, these cells were treated with canonical cell permeable peptides (Tat^(Dex) and Arg8^(Dex)) and miniature proteins containing a penta-Arg motif that reach the cytosol intact, with efficiencies exceeding 50% (5.3^(Dex) and ZF 5.3^(Dex)) (LaRochelle, J. R.; et al. Journal of the American Chemical Society 2015, 137, 2536). Aurein 1.2-+36 GFP^(Dex) (TR=2.62), activated significantly greater levels of GR-mCherry translocation (p<0.001) than Tat^(Dex) (TR=1.87) and Arg8^(Dex) (TR=1.63) and similar levels evoked by miniature proteins 5.3^(Dex) (TR=2.62) and ZF 5.3^(Dex) (TR=2.38) (FIGS. 17A and 17B). Taken together, these results evidence that aurein 1.2-+36 GFP^(Dex) exhibits an improved ability to access the cytoplasm over +36 GFP^(Dex) and canonical cell permeable peptides.

As an additional, independent assay of non-endosomal protein delivery, the ability of aurein 1.2 to enhance the nonendosomal delivery of an evolved biotin ligase (BirA) enzyme was tested (Howarth, M.; Ting, A. Y. Nature protocols 2008, 3, 534). BirA catalyzes the biotinylation of a 15-amino acid acceptor peptide (AP). A mCherry-AP fusion plasmid was transfected into HeLa cells. Biotinylation of mCherry can only occur in the presence of cytosolic BirA. To assess the non-endosomal delivery of +36 GFP-BirA protein, mCherry-AP biotinylation was quantified by Western blot using fluorophore-labeled streptavidin and normalized to the mCherry signal. Treatment with 250 nM aurein 1.2-+36 GFP-BirA resulted in a 50% increase in biotinylation signal compared with 250 nM of +36 GFP-BirA alone. A dose-dependent increase in AP-biotinylation across treatment concentrations (250 nM, 500 nM, and 1 μM) for both aurein 1.2-(+36GFP)-BirA and unfused +36 GFP-BirA constructs was observed. These results are consistent with the results of the GR translocation assay, and further evidence that aurein 1.2 enhances the endosomal escape of superpositively charged proteins.

In order to directly quantify the increase in non-endosomal delivery resulting from aurein 1.2, a cytosolic fractionation experiment was performed to calculate the cytosolic concentrations of delivered protein. HeLa cells were treated with +36 GFP or aurein 1.2-+36 GFP at 250 nM, 500 nM, and 1 μM. After 30 min of treatment, cells were washed, homogenized, and fractionated by ultracentrifugation. The cytosolic concentration of delivered protein was calculated from the GFP fluorescence of the cytosolic fraction together with a standard curve relating fluorescence to known concentrations of +36 GFP and aurein 1.2-+36 GFP added to cytosolic extract. At 250 nM, treatment with aurein 1.2-+36 GFP resulted in ˜5-fold more delivered cytosolic protein than treatment with +36 GFP alone. This difference decreased with increasing protein concentration, likely due to the influence of alternate uptake pathways or delivery bottlenecks at high protein concentrations. In contrast, the total amount of aurein 1.2-+36GFP versus +36 GFP taken up by cells was similar, with aurein 1.2-+36 GFP showing 1.3-fold higher total cellular uptake at 250 nM. These results directly demonstrate that aurein 1.2 increases the cytosolic concentration of cationic proteins that enter cells predominantly through endosomes (McNaughton, B. R.; et al. Proceedings of the National Academy of Sciences 2009, 106, 6111; Thompson, David B.; et al. Chemistry & Biology 2012, 19, 831) and are consistent with the above findings that aurein 1.2 has the greatest effect on enhancing non-endosomal delivery at ˜250 nM (FIG. 15A).

Effect of Endocytic Inhibitors on +36 GFP and Aurein 1.2-+36 GFP Delivery.

Endocytosis plays a key role in the cytosolic delivery of superpositively charged proteins (Thompson, David B.; et al. Chemistry & Biology 2012, 19, 831). To probe the role of endocytosis in the delivery of supercharged proteins with or without aurein 1.2, cells expressing GR-mCherry were treated with either +36 GFP^(Dex) or aurein 1.2-+36GFP^(Dex) in the presence of known endocytic inhibitors. The cortical actin remodeling inhibitor N-ethyl-isopropyl amiloride (EIPA), the cholesterol-sequestering agent methyl-β-cyclodextrin (MBCD), and the endosomal vesicular ATPase inhibitor bafilomycin (Baf) all strongly reduced the ability of both proteins to stimulate GRmCherry translocation. Blocking maturation of Rab5⁺ vesicles by treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin (Wort) did not influence reporter translocation of either +36 GFP^(Dex) or aurein 1.2-+36GFP^(Dex) (FIGS. 17C and 17D). In contrast, treatment with the small-molecule dynamin II inhibitor Dynasore (Dyna) significantly suppressed the ability of +36 GFP^(Dex) to stimulate GR-mCherry translocation (TR=1.64) (FIG. 17C) but had little influence on the cytosolic delivery of aurein 1.2-+36GFP^(Dex) (TR=2.30) (FIG. 17D). Taken together, these results evidence that active endocytosis is required for uptake of +36 GFP and aurein 1.2-+36GFP into the cell interior, and that the two proteins may traffic differently into the cell interior.

Aurein 1.2 can Greatly Increase Protein Delivery Efficiency In Vivo.

To evaluate the ability of aurein 1.2 to increase the efficacy of cationic protein delivery in vivo, proteins were delivered to the inner ear of Cre reporter transgenic mice that express tdTomato upon Cre-mediated recombination. This animal model was chosen due to its confined injection volume, the presence of well-characterized cell types, and the existence of genetic deafness models that would facilitate future studies of protein delivery to treat hearing loss. +36 GFP-Cre alone can be delivered to mouse retina (Cronican, J. J.; et al. ACS Chemical Biology 2010, 5, 747), albeit resulting in only modest levels of recombination consistent with inefficient endosomal escape.

Anesthetized postnatal day 2 (P2) mice were injected with 0.4 μL of 50 μM +36 GFP-Cre or aurein 1.2-+36 GFP-Cre solutions in the scala media to access the cochlear cells. Five days after injection, the cochleas were harvested for immunolabeling of inner ear cell markers and imaging for tdTomato florescence (FIG. 18A). Both the hair cells (Myo7a+) and supporting cells (Myo7a−) were evaluated for td Tomato signal. The total number of hair cells and supporting cells (by DAPI labeling) in the sensory epithelium (SE) was used to determine the relative toxicity of aurein 1.2-+36 GFP-Cre to the baseline treatment of +36 GFP-Cre (FIG. 18A). Overall, an average of 96%, 92% and 66% of cochlear cells survived aurein 1.2-+36 GFP-Cre treatment as compared to +36 GFP-Cretreatment in the apex, middle, and base tissue samples, respectively (FIG. 18A). +36 GFP-Cre treatment resulted in low levels of recombination only in inner hair cells (IHC) of the apex of the cochlea (4.4%) but not in the middle or base of the cochlear hair cells or any cochlear supporting cells. In contrast, treatment with aurein 1.2-+36 GFP-Cre resulted in very high Cre-mediated recombination levels throughout the apex, middle, and base samples of outer hair cells (OHC) (96%, 91%, and 69%, respectively), inner hair cells (100%, 94%, and 70%, respectively), as well as supporting cells (arrows) (FIGS. 18A and 18C).

The observed levels of recombination in the inner hair cells from aurein 1.2-+36 GFP-Cre are comparable to that of adeno-associated virus type 1 (AAV1) gene transfection (Akil, O.; et al. Neuron 2012, 75, 283). For outer hair cells, it similar levels of recombination was previously shown using liposome-mediated delivery of supernegatively-charged GFP-Cre (Zuris, J. A.; et al. Nat Biotech 2015, 33, 73). The aurein 1.2-+36 GFP-Cre delivery system is the only method that showed significant recombination levels in both inner and outer hair cells (Akil, O.; et al. Neuron 2012, 75, 283; Taura, A.; et al. Neuroscience 2010, 166, 1185), and does not require any virus or other molecules beyond a single polypeptide. Significantly, aurein 1.2-+36 GFP-Cre also extended delivered recombinase activity to additional cochlear supporting cells. These results evidence that the aurein 1.2-+36 GFP-Cre delivery system to be a promising method for in vivo protein delivery into both hair cells and supporting cells of the inner ear (Izumikawa, M.; et al. Nature Medicine 2005, 11, 271).

Discussion

A 13-residue peptide, aurein 1.2, was discovered that can increase the efficiency of non-endosomal protein delivery by screening a panel of known membrane-active peptides. The results from a small screen of 22 peptides are consistent with our hypothesis that some peptides can selectively disrupt the endosomal membrane without disrupting the mammalian cell membrane. The effectiveness of aurein 1.2 is highly dependent on its sequence, as several other closely related peptides did not enhance protein delivery (Tables 1 and 2). Subtle differences in amino acid composition led to dramatic changes in membrane activity among peptides tested, highlighting the difficulty of rationally designing peptides that enhance non-endosomal delivery. Moreover, the lack of correspondence between peptide cationic charge and non-endosomal delivery efficiency evidences that aurein 1.2 does not enhance non-endosomal delivery simply by promoting endocytosis. While none of the tested variants of aurein 1.2 substantially outperformed the original peptide, several amino acids were identified that could be altered without loss of activity. These findings also provide a starting point for further optimization to discover next-generation endosomolytic peptides with improved efficacy and reduced toxicity.

Four independent assays for non-endosomal protein delivery (Cre recombination, GR translocation, BirA activity on a cytoplasmic peptide, and cytosolic fractionation), together with the peptide mutational studies described above, collectively evidence that aurein 1.2-fusion enhances endosomal escape of superpositively charged proteins. Moreover, these assays collectively demonstrated the ability of aurein 1.2 to mediate the non-endosomal delivery of +36 GFP fused to different proteins (or small molecules), evidences that aurein 1.2 facilities endosomal escape.

The in vivo protein delivery experiments described above revealed dramatic increases in non-endosomal functional Cre recombinase delivery into the diverse inner ear cell types including hair cells and supporting cells of live mice upon fusion with aurein 1.2. Indeed, aurein 1.2-fused +36 GFP-Cre construct resulted in highly efficient recombination levels across the main cochlear sensory epithelial cell classes studied in this work, all but one of which were unaffected by +36 GFP-Cre treatment. Taken together, these results evidence that aurein 1.2 is a 13-residue, potent, genetically encodable, endosome escape-enhancing peptide that can greatly increase the efficiency of non-endosomal cationic protein delivery in vitro and in vivo without requiring the use of additional components beyond the protein of interest. 

1. A chimeric molecule comprising any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide, antimicrobial or membrane destabilizing peptide, or combinations thereof, and one or more gene editing agents comprising: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.
 2. The chimeric molecule of claim 1, wherein the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.
 3. A chimeric nucleic acid molecule encoding any two or more proteins, peptides or variants thereof, said proteins, peptides or variants thereof comprising: a supercharged protein or peptide, antimicrobial or membrane destabilizing domain, or combinations thereof, and one or more gene editing agents comprising: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.
 4. The chimeric molecule of claim 3, wherein the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.
 5. A chimeric molecule comprising an oligonucleotide or polynucleotide complexed, linked, or fused to one or more gene editing agents comprising: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.
 6. The chimeric molecule of claim 5, wherein the oligonucleotides or polynucleotides comprise: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, guide RNA (gRNA), modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), interference RNA, mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof.
 7. The chimeric molecule of claim 5, wherein the gene editing agents comprise: transcriptional activators, transcriptional repressors, recombinases, nucleases, DNA-binding proteins or nucleic acids, or combinations thereof.
 8. The chimeric molecule of claim 7, wherein the gene editing agents comprise: Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.
 9. The chimeric molecule of claim 5, further comprising a supercharged protein or peptide, antimicrobial or membrane destabilizing peptides or variants thereof.
 10. The chimeric molecule of claim 5, wherein the chimeric molecule is optionally encapsulated in a cationic lipid formulation.
 11. A chimeric molecule comprising at least one gene editing agent fused, complexed or linked to at least one guide RNA (gRNA).
 12. The chimeric molecule of claim 11, wherein the at least one gene editing agent comprises Cre recombinases, CRISPR/Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators or combinations thereof.
 13. The chimeric molecule of claim 11, optionally comprising a supercharged protein or peptide, antimicrobial or membrane destabilizing peptides, variants, or combinations thereof.
 14. The chimeric molecule of claim 11, wherein the chimeric molecule is optionally encapsulated in a cationic lipid formulation.
 15. (canceled) 